Antibodies For Oncogenic Strains Of HPV And Methods Of Their Use

ABSTRACT

The subject invention provides antibodies, including polyclonal and monoclonal antibodies, that bind to E6 proteins from at least three oncogenic strains of HPV. In general, the antibodies bind to amino acids motifs that are conserved between the E6 proteins of different HPV strains, particularly HPV strains 16 and 18. The subject antibodies may be used to detect HPV E6 protein in a sample, and, accordingly, the antibodies find use in a variety of diagnostic applications, including methods of diagnosing cancer. Kits for performing the subject methods and containing the subject antibodies are also provided.

CROSS-REFERENCE

This application is a continuation application of Ser. No. 12/660,613,filed Mar. 1, 2010, which is a continuation of Ser. No. 12/156,013,filed May 28, 2008, which is a continuation application of Ser. No.11/021,949, filed December 23 which claims the benefit of U.S.provisional patent application Ser. No. 60/532,373, filed Dec. 23, 2003,which applications are incorporated herein by reference in theirentirety.

FIELD OF INVENTION

The present invention relates to detection of oncogenic strains of humanpapillomavirus (HPV).

BACKGROUND OF THE INVENTION

Cervical cancer is the second most common cancer diagnosis in women andis linked to high-risk human papillomavirus infection 99.7% of the time.Currently, 12,000 new cases of invasive cervical cancer are diagnosed inUS women annually, resulting in 5,000 deaths each year. Furthermore,there are approximately 400,000 cases of cervical cancer and close to200,000 deaths annually worldwide. Human papillomaviruses (HPVs) are oneof the most common causes of sexually transmitted disease in the world.Overall, 50-75% of sexually active men and women acquire genital HPVinfections at some point in their lives. An estimated 5.5 million peoplebecome infected with HPV each year in the US alone, and at least 20million are currently infected. The more than 100 different isolates ofHPV have been broadly subdivided into high-risk and low-risk subtypesbased on their association with cervical carcinomas or with benigncervical lesions or dysplasias.

A number of lines of evidence point to HPV infections as the etiologicalagents of cervical cancers. Multiple studies in the 1980's reported thepresence of HPV variants in cervical dysplasias, cancer, and in celllines derived from cervical cancer. Further research demonstrated thatthe E6-E7 region of the genome from oncogenic HPV 18 is selectivelyretained in cervical cancer cells, suggesting that HPV infection couldbe causative and that continued expression of the E6-E7 region isrequired for maintenance of the immortalized or cancerous state. Furtherresearch demonstrated that the E6-E7 genes from HPV 16 were sufficientto immortalize human keratinocytes in culture. It was also demonstratedthat although E6-E7 genes from high risk HPVs could transform celllines, the E6-E7 regions from low risk, or non-oncogenic variants suchas HPV 6 and HPV 11 were unable to transform human keratinocytes. HPV 16and 18 infection was examined by in situ hybridization and E6 proteinexpression by immunocytochemistry in 623 cervical tissue samples atvarious stages of tumor progression and found a significant correlationbetween histological abnormality and HPV infection.

A significant unmet need exists for early and accurate diagnosis ofoncogenic HPV infection as well as for treatments directed at thecausative HPV infection, preventing the development of cervical cancerby intervening earlier in disease progression. Human papillomavirusescharacterized to date are associated with lesions confined to theepithelial layers of skin, or oral, pharyngeal, respiratory, and, mostimportantly, anogenital mucosae. Specific human papillomavirus types,including HPV 6 and 11, frequently cause benign mucosal lesions, whereasother types such as HPV 16, 18, and a host of other strains, arepredominantly found in high-grade lesions and cancer. Individual typesof human papillomaviruses (HPV) which infect mucosal surfaces have beenimplicated as the causative agents for carcinomas of the cervix, breast(Yu et al. (1999) Anticancer Res. 19:55555057-5061; Liu et al. (2001) J.Hum. Virol. 44:329-334), anus, penis, prostate (De Villiers et al.(1989) Virology 171:248:253), larynx and the buccal cavity, tonsils(Snijders et al. (1994) J. Gen. Virol. 75(Pt 10):2769-2775), nasalpassage (Trujillo et al. (1996) Virus Genes 12:165-178; Wu et al. (1993)Lancet 341:522-524), skin (Trenfield et al. (1993) Australas. J.Dermatol. 34:71-78), bladder (Baithun et al. (1998) Cancer Surv.31:17-27), head and neck squamous-cell carcinomas (Braakhuis et al.(2004) J. Natl. Cancer Inst. 96:978-980), occasional periungalcarcinomas, as well as benign anogenital warts. The identification ofparticular HPV types is used for identifying patients with premalignantlesions who are at risk of progression to malignancy. Although visibleanogenital lesions are present in some persons infected with humanpapillomavirus, the majority of individuals with HPV genital tractinfection do not have clinically apparent disease, but analysis ofcytomorphological traits present in cervical smears can be used todetect HPV infection. Papanicolaou tests are a valuable screening tool,but they miss a large proportion of HPV-infected persons due to theunfortunate false positive and false negative test results. In addition,they are not amenable to worldwide testing because interpretation ofresults requires trained pathologists.

HPV infection is also associated with Netherton's syndrome (Weber et al.(2001) Br. J. Dermatol. 144:1044-1049) and epidermolysis verruciformis(Rubaie et al. (1998) Int. J. Dermatol. 37:766-771). HPV can also betransmitted to a fetus by the mother (Smith et al. (2004) Sex. Transm.Dis. 31:57-62; Xu et al. (1998) Chin. Med.Sci. J. 13:29-31; Cason et al.(1998) Intervirology 41:213-218).

The detection and diagnosis of disease is a prerequisite for thetreatment of disease. Numerous markers and characteristics of diseaseshave been identified and many are used for the diagnosis of disease.Many diseases are preceded by, and are characterized by, changes in thestate of the affected cells. Changes can include the expression ofpathogengenes or proteins in infected cells, changes in the expressionpatterns of genes or proteins in affected cells, and changes in cellmorphology. The detection, diagnosis, and monitoring of diseases can beaided by the accurate assessment of these changes. Inexpensive, rapid,early and accurate detection of pathogens can allow treatment andprevention of diseases that range in effect from discomfort to death.

Literature

Literature of interest includes the following references: Zozulya etal., (Genome Biology 2:0018.1-0018.12, 2001; Mombairts (Annu. Rev.Neurosci 22:487-509, 1999); Raming et al., (Nature 361: 353-356, 1993);Belluscio et al., (Neuron 20: 69-81, 1988); Ronnet et al., (Annu. Rev.Physiol. 64:189-222, 2002); Lu et al., (Traffic 4: 416-533, 2003); Buck(Cell 100:611-618, 2000); Malnic et al., (Cell 96:713-723, 1999);Firestein (Nature 413:211-218, 2001); Zhao et al., (Science 279:237-242, 1998); Touhara et al., (Proc. Natl. Acad. Sci. 96: 4040-4045,1999); Sklar et al., (J. Biol. Chem. 261:15538-15543, 1986); Dryer etal., (TiPS 20:413-417, 1999); Ivic et al., (J. Neurobiol. 50:56-68,2002); Munger (2002) Front. Biosci. 7:d641-9; Glaunsinger (2000)Oncogene 19:5270-80; Gardiol (1999) Oncogene 18:5487-96; Pim (1999)Oncogene 18:7403-8; Meschede (1998) J. Clin. Microbiol. 36:475-80;Kiyono (1997) Proc. Natl. Acad. Sci. 94:11612-6; and Lee (1997) Proc.Natl. Acad. Sci. 94:6670-5; Banks (1987) J. Gen. Virol. 68:1351-1359;Fuchs et al., (Hum. Genet. 108:1-13, 2001); and Giovane et al. (1999)Journal of Molecular Recognition 12:141-152 and published US patentapplications 20030143679 and 20030105285; and U.S. Pat. Nos. 6,610,511,6,492,143 6,410,249, 6,322,794, 6,344,314, 5,415,995, 5,753,233,5,876,723, 5,648,459, 6,391,539, 5,665,535 and 4,777,239.

SUMMARY OF THE INVENTION

The subject invention provides antibodies, including polyclonal andmonoclonal antibodies, that bind to the E6 proteins from at least threedifferent oncogenic strains of HPV. In general, the antibodies bind toamino acids motifs that are conserved between the E6 proteins ofdifferent HPV strains, particularly HPV strains 16 and 18. The subjectantibodies may be used to detect HPV E6 protein in a sample, and,accordingly, the antibodies find use in a variety of diagnosticapplications, including methods of diagnosing cancer. Kits forperforming the subject methods and containing the subject antibodies arealso provided.

In certain embodiments, the invention provides an antibody compositioncomprising a monoclonal antibody that specifically binds to the E6proteins of at least three (e.g., 4, 5, 6, 7 or 8 or more, usually up to10 or 12) different oncogenic HPV strains. The antibody composition maycomprise a mixture of monoclonal antibodies that specifically bind tothe E6 proteins of HPV strains 16, 18, 31, 33 and 45 (e.g., HPV strains16, 18, 31, 33, 45, 52 and 58, HPV strains 16, 18, 31, 33, 45, 52, 58,35 and 59 or HPV strains 16, 18, 26, 30, 31, 33, 34, 45, 51, 52, 53, 58,59, 66, 68b, 69, 70, 73 and 82, wherein at least one of said monoclonalantibodies specifically binds to the E6 proteins of at least threedifferent oncogenic HPV strains. In certain embodiments, the monoclonalantibody may bind to the E6 proteins of HPV strains 16 and 18, whereinsaid antibody binds SEQ ID NOS: 1, 3 or 5 of HPV strain 16 E6 and SEQ IDNOS: 2, 4 or 6 of HPV strain 18 E6. In certain embodiments, themonoclonal antibody binds to E6 proteins of HPV strains 16 and 45 or HPVstrains 16, 18, 31, 33 and 45.

The invention also provides a method of detecting an HPV E6 protein in asample. This methods generally involves contacting the subject antibodycomposition with the sample, and detecting any binding of the antibodiesin the composition to the sample, wherein binding of an antibody to thesample indicates the presence of an HPV E6 protein. The sample may besuspected of containing an oncogenic strain of HPV.

The invention also provides a system for detecting the presence of anoncogenic HPV E6 polypeptide in a sample. This system generallycomprises a first and a second binding partner for an oncogenic HPV E6polypeptide, wherein the first binding partner is a PDZ domain proteinand said second binding partner is an subject antibody. At least one ofsaid binding partners is attached to a solid support.

The invention also provides a method of detecting the presence of anoncogenic HPV E6 protein in a sample. This method generally comprises:contacting a sample containing an oncogenic HPV E6 protein with a PDZdomain polypeptide; and detecting any binding of the oncogenic HPV E6protein in said sample to said PDZ domain polypeptide using an subjectantibody, wherein binding of the oncogenic HPV E6 protein to said PDZdomain polypeptide indicates the presence of an oncogenic HPV E6 proteinin said sample.

The invention also provides a kit containing a subject antibody; andinstructions for using the antibody to detect a HPV E6 protein. The kitmay also contain a PDZ domain polypeptide.

The invention also provides a peptide of less than 15 amino acidscomprising any one of the sequences set forth in Table 1.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is an alignment of E6 amino acid sequences from various oncogenicstrains of HPV. From top to bottom, the various HPV E6 amino acidsequences are set forth in the sequence listing as SEQ ID NOS: 13-32,respectively. Amino acid sequence from three other oncogenic strains ofHPV (strains 34, 67 and 70) are found in the sequence listing as SEQ IDNOS: 359-361.

FIG. 2 is an alignment of E6 amino acid sequences from a subset ofoncogenic strains of HPV shown in FIG. 1.

FIG. 3 is a slot western blot showing antibody reactivity with E6protein.

FIG. 4 is graph showing cross-reactivity of F22-10D11 monoclonalantibody.

DEFINITIONS

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may of course vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Throughout this application, various publications, patents and publishedpatent applications are cited. The disclosures of these publications,patents and published patent applications referenced in this applicationare hereby incorporated by reference in their entirety into the presentdisclosure. Citation herein by Applicant of a publication, patent, orpublished patent application is not an admission by Applicant of saidpublication, patent, or published patent application as prior art.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “asample” includes a plurality of such sample, and reference to “theantibody” includes reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth. It is furthernoted that the claims may be drafted to exclude any optional element. Assuch, this statement is intended to serve as antecedent basis for use ofsuch exclusive terminology as “solely”, “only” and the like inconnection with the recitation of claim elements, or the use of a“negative” limitation.

A “biopolymer” is a polymer of one or more types of repeating units,regardless of the source. Biopolymers may be found in biological systemsand particularly include polypeptides and polynucleotides, as well assuch compounds containing amino acids, nucleotides, or analogs thereof.The term “polynucleotide” refers to a polymer of nucleotides, or analogsthereof, of any length, including oligonucleotides that range from10-100 nucleotides in length and polynucleotides of greater than 100nucleotides in length. The term “polypeptide” refers to a polymer ofamino acids of any length, including peptides that range from 6-50 aminoacids in length and polypeptides that are greater than about 50 aminoacids in length.

In most embodiments, the terms “polypeptide” and “protein” are usedinterchangeably. The term “polypeptide” includes polypeptides in whichthe conventional backbone has been replaced with non-naturally occurringor synthetic backbones, and peptides in which one or more of theconventional amino acids have been replaced with a non-naturallyoccurring or synthetic amino acid capable of participating in peptidebonding interactions. The term “fusion protein” or grammaticalequivalents thereof is meant a protein composed of a plurality ofpolypeptide components, that while typically not attached in theirnative state, typically are joined by their respective amino andcarboxyl termini through a peptide linkage to form a single continuouspolypeptide. Fusion proteins may be a combination of two, three or evenfour or more different proteins. The term polypeptide includes fusionproteins, including, but not limited to, fusion proteins with aheterologous amino acid sequence, fusions with heterologous andhomologous leader sequences, with or without N-terminal methionineresidues; immunologically tagged proteins; fusion proteins withdetectable fusion partners, e.g., fusion proteins including as a fusionpartner a fluorescent protein, β-galactosidase, luciferase, etc., andthe like.

In general, polypeptides may be of any length, e.g., greater than 2amino acids, greater than 4 amino acids, greater than about 10 aminoacids, greater than about 20 amino acids, greater than about 50 aminoacids, greater than about 100 amino acids, greater than about 300 aminoacids, usually up to about 500 or 1000 or more amino acids. “Peptides”are generally greater than 2 amino acids, greater than 4 amino acids,greater than about 10 amino acids, greater than about 20 amino acids,usually up to about 50 amino acids. In some embodiments, peptides arebetween 5 and 30 or between 8 and 15 amino acids in length.

The term “capture agent” refers to an agent that binds an analytethrough an interaction that is sufficient to permit the agent to bindand concentrate the analyte from a homogeneous mixture of differentanalytes. The binding interaction is typically mediated by an affinityregion of the capture agent. Typical capture agents include anypolypeptide, e.g., a PDZ protein, however antibodies may be employed.Capture agents usually “specifically bind” one or more analytes, e.g.,an oncogenic E6 protein. Accordingly, the term “capture agent” refers toa molecule or a multi-molecular complex which can specifically bind ananalyte, e.g., specifically bind an analyte for the capture agent, witha dissociation constant (K_(D)) of less than about 10⁻⁶ M withoutbinding to other targets.

The term “specific binding” refers to the ability of a capture agent topreferentially bind to a particular analyte that is present in ahomogeneous mixture of different analytes. Typically, a specific bindinginteraction will discriminate between desirable and undesirable analytesin a sample, typically more than about 10 to 100-fold or more (e.g.,more than about 1000- or 10,000-fold). Typically, the affinity between acapture agent and analyte when they are specifically bound in a captureagent/analyte complex is at least 10⁻⁷, at least 10⁻⁸ M, at least 10⁻⁹M, usually up to about 10⁻¹⁰ M.

The term “capture agent/analyte complex” is a complex that results fromthe specific binding of a capture agent with an analyte, i.e., a“binding partner pair”. A capture agent and an analyte for the captureagent will typically specifically bind to each other under “conditionssuitable to for specific binding”, where such conditions are thoseconditions (in terms of salt concentration, pH, detergent, proteinconcentration, temperature, etc.) which allow for binding to occurbetween capture agents and analytes to bind in solution. Suchconditions, particularly with respect to antibodies and their antigens,are well known in the art (see, e.g., Harlow and Lane (Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1989)). Conditions suitable for specific binding typically permitcapture agents and target pairs that have a dissociation constant(K_(D)) of less than about 10⁻⁶ M to bind to each other, but not withother capture agents or targets.

As used herein, “binding partners” and equivalents refer to pairs ofmolecules that can be found in a capture agent/analyte complex, i.e.,exhibit specific binding with each other.

The phrase “surface-bound capture agent” refers to a capture agent thatis immobilized on a surface of a solid substrate, where the substratecan have a variety of configurations, e.g., a sheet, bead, strip, orother structure, such as a plate with wells.

The term “pre-determined” refers to an element whose identity is knownprior to its use. For example, a “pre-determined analyte” is an analytewhose identity is known prior to any binding to a capture agent. Anelement may be known by name, sequence, molecular weight, its function,or any other attribute or identifier. In some embodiments, the term“analyte of interest”, i.e., an known analyte that is of interest, isused synonymously with the term “pre-determined analyte”.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein to refer to a type capture agent that has at least an epitopebinding domain of an antibody. These terms are well understood by thosein the field, and refer to a protein containing one or more polypeptidesthat specifically binds an antigen. One form of antibody constitutes thebasic structural unit of an antibody. This form is a tetramer andconsists of two identical pairs of antibody chains, each pair having onelight and one heavy chain. In each pair, the light and heavy chainvariable regions are together responsible for binding to an antigen, andthe constant regions are responsible for the antibody effectorfunctions.

The recognized immunoglobulin polypeptides include the kappa and lambdalight chains and the alpha, gamma (IgG₁, IgG₂, IgG₃, IgG₄), delta,epsilon and mu heavy chains or equivalents in other species. Full-lengthimmunoglobulin “light chains” (of about 25 kDa or about 214 amino acids)comprise a variable region of about 110 amino acids at the NH₂-terminusand a kappa or lambda constant region at the COOH-terminus. Full-lengthimmunoglobulin “heavy chains” (of about 50 kDa or about 446 aminoacids), similarly comprise a variable region (of about 116 amino acids)and one of the aforementioned heavy chain constant regions, e.g., gamma(of about 330 amino acids).

The terms “antibodies” and “immunoglobulin” include antibodies orimmunoglobulins of any isotype, fragments of antibodies which retainspecific binding to antigen, including, but not limited to, Fab, Fv,scFv, and Fd fragments, chimeric antibodies, humanized antibodies,single-chain antibodies, and fusion proteins comprising anantigen-binding portion of an antibody and a non-antibody protein. Theantibodies may be detectably labeled, e.g., with a radioisotope, anenzyme which generates a detectable product, a fluorescent protein, andthe like. The antibodies may be further conjugated to other moieties,such as members of specific binding pairs, e.g., biotin (member ofbiotin-avidin specific binding pair), and the like. The antibodies mayalso be bound to a solid support, including, but not limited to,polystyrene plates or beads, and the like. Also encompassed by the termsare Fab′, Fv, F(ab′)₂, and or other antibody fragments that retainspecific binding to antigen.

Antibodies may exist in a variety of other forms including, for example,Fv, Fab, and (Fab′)₂, as well as bi-functional (i.e. bi-specific) hybridantibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987))and in single chains (e.g., Huston et al., Proc. Natl. Acad. Sci.U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426(1988), which are incorporated herein by reference). (See, generally,Hood et al, Immunology, Benjamin, N.Y., 2nd ed. (1984), and Hunkapillerand Hood, Nature, 323, 15-16 (1986). Monoclonal antibodies, polyclonalantibodies, and “phage display” antibodies are well known in the art andencompassed by the term “antibodies”.

The term “mixture”, as used herein, refers to a combination of elements,e.g., capture agents or analytes, that are interspersed and not in anyparticular order. A mixture is homogeneous and not spatially separableinto its different constituents. Examples of mixtures of elementsinclude a number of different elements that are dissolved in the sameaqueous solution, or a number of different elements attached to a solidsupport at random or in no particular order in which the differentelements are not specially distinct. In other words, a mixture is notaddressable. To be specific, an array of capture agents, as is commonlyknown in the art, is not a mixture of capture agents because the speciesof capture agents are spatially distinct and the array is addressable.

“Isolated” or “purified” general refers to isolation of a substance(compound, polynucleotide, protein, polypeptide, polypeptidecomposition) such that the substance comprises the majority percent ofthe sample in which it resides. Typically in a sample a substantiallypurified component comprises 50%, preferably 80%-85%, more preferably90-95% of the sample. Techniques for purifying polynucleotides andpolypeptides, e.g., antibodies, of interest are well-known in the artand include, for example, ion-exchange chromatography, affinitychromatography and sedimentation according to density.

The term “assessing” refers to any form of measurement, and includesdetermining if an element is present or not. The terms “determining”,“measuring”, “evaluating”, “assessing” and “assaying” are usedinterchangeably and include both quantitative and qualitativedeterminations. Assessing may be relative or absolute. “Assessing thepresence of includes determining the amount of something present, aswell as determining whether it is present or absent.

The term ‘marker” or “biological marker”, as used herein, refers to ameasurable or detectable entity in a biological sample. Examples ormarkers include nucleic acids, proteins, or chemicals that are presentin biological samples. One example of a marker is the presence of viralor pathogen proteins or nucleic acids in a biological sample from ahuman source.

The term “sample” as used herein relates to a material or mixture ofmaterials, typically, although not necessarily, in fluid form, i.e.,aqueous, containing one or more components of interest. Samples may bederived from a variety of sources such as from food stuffs,environmental materials, a biological sample or solid, such as tissue orfluid isolated from an individual, including but not limited to, forexample, plasma, serum, spinal fluid, semen, lymph fluid, the externalsections of the skin, respiratory, intestinal, and genitourinary tracts,tears, saliva, milk, blood cells, tumors, organs, and also samples of invitro cell culture constituents (including but not limited toconditioned medium resulting from the growth of cells in cell culturemedium, putatively virally infected cells, recombinant cells, and cellcomponents). The term “biological sample” is meant to distinguishbetween a sample in a clinical setting from a sample that may be arecombinant sample or derived from a recombinant sample.

Components in a sample are termed “analytes” herein. In manyembodiments, the sample is a complex sample containing at least about10², 5×10², 10³, 5×10³, 10⁴, 5×10⁴, 10⁵, 5×10⁵, 10⁶, 5×10⁶, 10⁷, 5×10⁷,10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹² or more species of analyte.

The term “analyte” is used herein interchangeably and refers to a knownor unknown component of a sample, which will specifically bind to acapture agent if the analyte and the capture agent are members of aspecific binding pair. In general, analytes are biopolymers, i.e., anoligomer or polymer such as an oligonucleotide, a peptide, apolypeptide, an antibody, or the like. In this case, an “analyte” isreferenced as a moiety in a mobile phase (typically fluid), to bedetected by a “capture agent” which, in some embodiments, is bound to asubstrate, or in other embodiments, is in solution. However, either ofthe “analyte” or “capture agent” may be the one which is to be evaluatedby the other (thus, either one could be an unknown mixture of analytes,e.g., polypeptides, to be evaluated by binding with the other).

A “fusion protein” or “fusion polypeptide” as used herein refers to acomposite protein, i.e., a single contiguous amino acid sequence, madeup of two (or more) distinct, heterologous polypeptides that are notnormally fused together in a single amino acid sequence. Thus, a fusionprotein can include a single amino acid sequence that contains twoentirely distinct amino acid sequences or two similar or identicalpolypeptide sequences, provided that these sequences are not normallyfound together in the same configuration in a single amino acid sequencefound in nature. Fusion proteins can generally be prepared using eitherrecombinant nucleic acid methods, i.e., as a result of transcription andtranslation of a recombinant gene fusion product, which fusion comprisesa segment encoding a polypeptide of the invention and a segment encodinga heterologous protein, or by chemical synthesis methods well known inthe art.

An “oncogenic HPV strain” is an HPV strain that is known to causecervical cancer as determined by the National Cancer Institute (NCI,2001). “Oncogenic E6 proteins” are E6 proteins encoded by the aboveoncogenic HPV strains. The sequences of exemplary oncogenic E6 proteinsof interest are shown in FIG. 1. The sequences of various HPV proteinsare also found as database entries at NCBI's Genbank database, asfollows: HPV16-E6: GI:9627100; HPV18-E6: GI:9626069; HPV31-E6:GI:9627109; HPV35-E6: GI:9627127; HPV30-E6: GI:9627320; HPV39-E6:GI:9627165; HPV45-E6: GI:9627356; HPV51-E6: GI:9627155; HPV52-E6:GI:9627370; HPV56-E6: GI:9627383; HPV59-E6: GI:9627962; HPV58-E6:GI:9626489; HPV33-E6: GI:9627118; HPV66-E6: GI:9628582; HPV68b-E6:GI:I84383; HPV69-E6: GI:9634605; HPV26-E6: GI:396956; HPV53-E6:GI:9627377; HPV73: GI:1491692; HPV82: GI:9634614, HPV34 GI:396989; HPV67GI:3228267; and HPV70 GI:1173493.

An “oncogenic E6 protein binding partner” can be any molecule thatspecifically binds to an oncogenic E6 protein. Suitable oncogenic E6protein binding partners include a PDZ domain (as described below),antibodies against oncogenic E6 proteins (such as those describedbelow); other proteins that recognize oncogenic E6 protein (e.g., p53,E6-AP or E6-BP); DNA (i.e., cruciform DNA); and other partners such asaptamers. In some embodiments, detection of more than 1 oncogenic E6protein (e.g., all oncogenic E6 proteins, E6 proteins from HPV strains16 and 18, or E6 proteins from HPV strains 16 and 45 etc.) is desirable,and, as such, an oncogenic E6 protein binding partner may be antibodythat binds to these proteins, as described below, or a mixture ofantibodies that each bind to a different proteins. As is known in theart, such binding partners may be labeled to facilitate their detection.In general, binding partner bind E6 with an binding affinity of lessthen 10⁻⁵ M, e.g., less than 10⁻⁶, less than 10⁻⁷, less than 10⁻⁸ M(e.g., less than 10⁻⁹ M, 10⁻¹⁰, 10⁻¹¹, etc.).

As used herein, the term “PDZ domain” refers to protein sequence of lessthan approximately 90 amino acids, (i.e., about 80-90, about 70-80,about 60-70 or about 50-60 amino acids), characterized by homology tothe brain synaptic protein PSD-95, the Drosophila septate junctionprotein Discs-Large (DLG), and the epithelial tight junction protein ZO1(ZO1). PDZ domains are also known as Discs-Large homology repeats(“DHRs”) and GLGF repeats. PDZ domains generally appear to maintain acore consensus sequence (Doyle, D. A., 1996, Cell 85: 1067-76).

PDZ domains are found in diverse membrane-associated proteins includingmembers of the MAGUK family of guanylate kinase homologs, severalprotein phosphatases and kinases, neuronal nitric oxide synthase, tumorsuppressor proteins, and several dystrophin-associated proteins,collectively known as syntrophins.

Exemplary PDZ domain-containing proteins and PDZ domain sequences areshown in TABLE 2. The term “PDZ domain” also encompasses variants (e.g.,naturally occurring variants) of the sequences (e.g., polymorphicvariants, variants with conservative substitutions, and the like) anddomains from alternative species (e.g. mouse, rat). Typically, PDZdomains are substantially identical to those shown in U.S. patentapplication Ser. Nos. 09/724,553 and 10/938,249), e.g., at least about70%, at least about 80%, or at least about 90% amino acid residueidentity when compared and aligned for maximum correspondence. It isappreciated in the art that PDZ domains can be mutated to give aminoacid changes that can strengthen or weaken binding and to alterspecificity, yet they remain PDZ domains (Schneider et al., 1998, Nat.Biotech. 17:170-5). Unless otherwise indicated, a reference to aparticular PDZ domain (e.g. a MAGI-1 domain 2) is intended to encompassthe particular PDZ domain and HPV E6-binding variants thereof. In otherwords, if a reference is made to a particular PDZ domain, a reference isalso made to variants of that PDZ domain that bind oncogenic E6 proteinof HPV, as described below. In this respect it is noted that thenumbering of PDZ domains in a protein may change. For example, theMAGI-1 domain 2, as referenced herein, may be referenced as MAGI-1domain 1 in other literature. As such, when a particular PDZ domain of aprotein is referenced in this application, this reference should beunderstood in view of the sequence of that domain, as described herein,particularly in the sequence listing. Table 2 shows the relationshipbetween the sequences of the sequence listing and the names and Genbankaccession numbers for various domains, where appropriate. Furtherdescription of PDZ proteins, particularly a description of MAGI-1 domain2 protein, is found in Ser. No. 10/630,590, filed Jul. 29, 2003 andpublished as US20040018487. This publication is incorporated byreference herein in its entirety for all purposes.

As used herein, the term “PDZ protein” refers to a naturally occurringprotein containing a PDZ domain. Exemplary PDZ proteins include CASK,MPP1, DLG1, DLG2, PSD95, NeDLG, TIP-33, SYN1a, TIP-43, LDP, LIM, LIMK1,LIMK2, MPP2, NOS1, AF6, PTN-4, prIL16, 41.81kD, KIAA0559, RGS12,KIAA0316, DVL1, TIP-40, TIAM1, MINT1, MAGI-1, MAGI-2, MAGI-3, KIAA0303,CBP, MINT3, TIP-2, KIAA0561, and TIP-1.

As used herein, the term “PL protein” or “PDZ Ligand protein” refers toa protein that forms a molecular complex with a PDZ-domain, or to aprotein whose carboxy-terminus, when expressed separately from the fulllength protein (e.g., as a peptide fragment of 4-25 residues, e.g., 8,10, 12, 14 or 16 residues), forms such a molecular complex. Themolecular complex can be observed in vitro using a variety of assaysdescribed infra.

As used herein, a “PL sequence” refers to the amino acid sequence of theC-terminus of a PL protein (e.g., the C-terminal 2, 3, 4, 5, 6, 7, 8, 9,10, 12, 14, 16, 20 or 25 residues) (“C-terminal PL sequence”) or to aninternal sequence known to bind a PDZ domain (“internal PL sequence”).

As used herein, a “PL fusion protein” is a fusion protein that has a PLsequence as one domain, typically as the C-terminal domain of the fusionprotein. An exemplary PL fusion protein is a tat-PL sequence fusion.

In the case of the PDZ domains described herein, a “HPV E6-bindingvariant” of a particular PDZ domain is a PDZ domain variant that retainsHPV E6 PDZ ligand binding activity. Assays for determining whether a PDZdomain variant binds HPV E6 are described in great detail below, andguidance for identifying which amino acids to change in a specific PDZdomain to make it into a variant may be found in a variety of sources.In one example, a PDZ domain may be compared to other PDZ domainsdescribed herein and amino acids at corresponding positions may besubstituted, for example. In another example, the sequence a PDZ domainof a particular PDZ protein may be compared to the sequence of anequivalent PDZ domain in an equivalent PDZ protein from another species.For example, the sequence of a PDZ domain from a human PDZ protein maybe compared to the sequence of other known and equivalent PDZ domainsfrom other species (e.g., mouse, rat, etc.) and any amino acids that arevariant between the two sequences may be substituted into the human PDZdomain to make a variant of the PDZ domain. For example, the sequence ofthe human MAGI-1 PDZ domain 2 may be compared to equivalent MAGI-1 PDZdomains from other species (e.g. mouse Genbank gi numbers 7513782 and28526157 or other homologous sequences) to identify amino acids that maybe substituted into the human MAGI-1-PDZ domain to make a variantthereof. Such method may be applied to any of the MAGI-1 PDZ domainsdescribed herein. Minimal MAGI-PDZ domain 2 sequence is provided as SEQID NOS:68-76. Particular variants may have 1, up to 5, up to about 10,up to about 15, up to about 20 or up to about 30 or more, usually up toabout 50 amino acid changes as compared to a sequence set forth in thesequence listing. Exemplary MAGI-1 PDZ variants include the sequencesset forth in SEQ ID NOS: 76-105. In making a variant, if a GFG motif ispresent in a PDZ domain, in general, it should not be altered insequence.

In general, variant PDZ domain polypeptides have a PDZ domain that hasat least about 70 or 80%, usually at least about 90%, and more usuallyat least about 98% sequence identity with a variant PDZ domainpolypeptide described herein, as measured by BLAST 2.0 using defaultparameters, over a region extending over the entire PDZ domain.

As used herein, a “detectable label” has the ordinary meaning in the artand refers to an atom (e.g., radionuclide), molecule (e.g.,fluorescein), or complex, that is or can be used to detect (e.g., due toa physical or chemical property), indicate the presence of a molecule orto enable binding of another molecule to which it is covalently bound orotherwise associated. The term “label” also refers to covalently boundor otherwise associated molecules (e.g., a biomolecule such as anenzyme) that act on a substrate to produce a detectable atom, moleculeor complex. Detectable labels suitable for use in the present inventioninclude any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Labels useful in the present invention include biotin for staining withlabeled streptavidin conjugate, magnetic beads (e.g., Dynabeads™),fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, greenfluorescent protein, enhanced green fluorescent protein, and the like),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g.,hydrolases, particularly phosphatases such as alkaline phosphatase,esterases and glycosidases, or oxidoreductases, particularly peroxidasessuch as horse radish peroxidase, and others commonly used in ELISAs),substrates, cofactors, inhibitors, chemiluminescent groups, chromogenicagents, and colorimetric labels such as colloidal gold or colored glassor plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.Patents disclosing such labels include U.S. Pat. Nos. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.Means of detecting such labels are well known to those of skill in theart.

As used herein, the terms “sandwich”, “sandwich ELISA”, “Sandwichdiagnostic” and “capture ELISA” all refer to the concept of detecting abiological polypeptide with two different test agents. For example, aPDZ protein could be directly or indirectly attached to a solid support.Test sample could be passed over the surface and the PDZ protein couldbind its cognate PL protein(s). A labeled antibody or alternativedetection reagent could then be used to determine whether a specific PLprotein had bound the PDZ protein.

By “solid phase support” or “carrier” is intended any support capable ofbinding polypeptide, antigen or antibody. Well-known supports orcarriers, include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses, and magnetite. The nature of the carrier canbe either soluble to some extent or insoluble for the purposes of thepresent invention. The support material can have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to a PDZ domain polypeptide or an E6 antibody. Thus, the supportconfiguration can be spherical, as in a bead, or cylindrical, as in theinside surface of a test tube, or the external surface of a rod.Alternatively, the surface can be flat, such as a sheet, culture dish,test strip, etc. Those skilled in the art will know many other suitablecarriers for binding antibody, peptide or antigen, or can ascertain thesame by routine experimentation.

In some embodiments “proteasome inhibitors”, i.e., inhibitors of theproteasome, may be used. These inhibitors, includingcarbobenzoxyl-leucinyl-leuciny-1 norvalinal II (MG 115) or CBZ-LLL, canbe purchased from chemical supply companies (e.g., Sigma). As a skilledperson would understand, proteasome inhibitors are not proteaseinhibitors.

As used herein, a “plurality” of components has its usual meaning. Insome embodiments, the plurality is at least 5, and often at least 25, atleast 40, or at least 60 or more, usually up to about 100 or 1000.

Reference to an “amount” of a E6 protein in these contexts is notintended to require quantitative assessment, and may be eitherqualitative or quantitative, unless specifically indicated otherwise.

The term “non-naturally occurring” or “recombinant” means artificial orotherwise not found in nature. Recombinant cells usually contain nucleicacid that is not usually found in that cell, recombinant nucleic acidusually contain a fusion of two or more nucleic acids that is not foundin nature, and a recombinant polypeptide is usually produced by arecombinant nucleic acid.

“Subject”, “individual,” “host” and “patient” are used interchangeablyherein, to refer to any animal, e.g., mammal, human or non-human.Generally, the subject is a mammalian subject. Exemplary subjectsinclude, but are not necessarily limited to, humans, non-human primates,mice, rats, cattle, sheep, goats, pigs, dogs, cats, birds, deer, elk,rabbit, reindeer, deer, and horses, with humans being of particularinterest.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

The subject invention provides antibodies, including polyclonal andmonoclonal antibodies, that bind to E6 proteins from at least threeoncogenic strains of HPV. In general, the antibodies bind to amino acidmotifs that are conserved between the E6 proteins of different HPVstrains, particularly HPV strains 16 and 18. The subject antibodies maybe used to detect HPV E6 protein in a sample, and, accordingly, theantibodies find use in a variety of diagnostic applications, includingmethods of diagnosing cancer. Kits for performing the subject methodsand containing the subject antibodies are also provided.

In further describing the invention in greater detail than provided inthe Summary and as informed by the Background and Definitions providedabove, the subject antibodies are described first, followed by adescription of methods in which the subject antibodies find use.Finally, kits for performing the subject methods are described.

Antibody Compositions

The invention provides antibodies, particularly monoclonal antibodies,that bind to E6 proteins of multiple strains of HPV. In other words, theinvention provides antibodies that “recognize”, i.e., specifically bindto with K_(D) of 10⁻⁶ M or less, multiple E6 proteins. In other words,the subject antibodies each bind to (i.e., cross-react with) a pluralityof different E6 proteins (i.e., at least 2, at least 3, at least 4, atleast 5, at least 6 or at least 10, usually up to about 12, 15 or 20 ormore different E6 proteins) from oncogenic, and, in certain embodiments,non-oncogenic strains of HPV. In general, the subject antibodies bind toamino acid motifs that are conserved between the E6 proteins ofdifferent HPV strains, and, accordingly, bind to E6 proteins that havethis motif. In many embodiments the antibodies bind at least the E6proteins of HPV strains 16 and 18 (e.g. the E6 proteins of HPV strains16, 18, 31, 33 and 45; 16, 18 and 45; or, in other embodiments, the E6proteins of all of the HPV strains listed in FIG. 1 or 2). In otherembodiments, the antibodies bind to at least the E6 proteins from HPVstrains 16 and 45. The subject antibodies may bind E6 protein fromnon-oncogenic strains of HPV (e.g., HPV strains 6 and/or 11) and,accordingly, the subject antibodies may bind to E6 proteins fromoncogenic, as well as non-oncogenic, strains of HPV.

The subject antibodies may specifically bind to one of three sequencemotifs found in HPV E6 proteins. These motifs are boxed in FIG. 1, andgenerally correspond to regions of sequence similarity between E6proteins from different strains of HPV. In general, therefore, a subjectantibody binds to peptides having the following sequence:FQDPQERPRKLPQLCTELQTTIHDI (SEQ ID NO:1) and FEDPTRRPYKLPDLCTELNTSLQDI(SEQ ID NO:2), corresponding to a first common sequence motif in the E6proteins of HPV strains 16 and 18, respectively, LLIRCINCQKPLCPEEKQRHLDK(SEQ ID NO:3) and LLIRCLRCQKPLNPAEKLRHLNE (SEQ ID NO:4), correspondingto a second common sequence motif in the E6 proteins of HPV strains 16and 18, respectively, or RHLDKKQRFHNIRGRWTGRCMSCC (SEQ ID NO:5) andRHLNEKRRFHNIAGHYRGQCHSCC (SEQ ID NO:6) corresponding to a third commonsequence motif in the E6 proteins of HPV strains 16 and 18,respectively. If a subject antibody binds to other E6 proteins, then itusually binds to the other E6 proteins at positions equivalent to thosediscussed above, or boxed in FIG. 1, where “positions equivalent to”generally means a stretch of contiguous amino acids that correspond to,i.e., are aligned with, the boxed amino acids when the sequence of theother E6 proteins are with those in FIG. 1.

Accordingly, since antibodies generally recognize motifs smaller thanthose listed above, a subject antibody may recognize peptides that aresmaller than and contained within the motifs described above. Forexample, a subject antibody may bind to a peptide having any 9contiguous amino acids set forth in any one of SEQ NOS:1-6. Inparticular, a subject antibody may recognize the sequences RPRKLPQLCTEL(SEQ ID NO:7) and RPYKLPDLCTEL (SEQ ID NO:8), corresponding tosub-sequences of the first common sequences of E6 proteins of HPVstrains 16 and 18, described above, LLIRCINCQKPL (SEQ ID NO:9) andLLIRCLRCQKPL (SEQ ID NO:10) corresponding to sub-sequences of the secondcommon sequences of E6 proteins of HPV strains 16 and 18, as describedabove, or RHLDKKQRFHNI (SEQ ID NO:11) and RHLNEKRRFHNI (SEQ ID NO:12)corresponding to sub-sequences of the third common sequences of E6proteins of HPV strains 16 and 18, as described above. Since thesesub-sequences are generally conserved between different E6 proteins, asdiscussed above, antibodies that bind to the above-recited sequencesgenerally bind to E6 proteins from other HPV strains.

In certain alternative embodiments, the subject antibodies will bind toE6 proteins from HPV strains 16 and 45. In general, therefore, a subjectantibody binds to peptides having the following sequence:FQDPQERPRKLPQLCTELQTTIHDI (SEQ ID NO:1) and FDDPKQRPYKLPDLCTELNTSLQDV(SEQ ID NO:57), corresponding to a first common sequence motif in the E6proteins of HPV strains 16 and 45, respectively, LLIRCINCQKPLCPEEKQRHLDK(SEQ ID NO:3) and LLIRCLRCQKPLNPAEKRRHLKD (SEQ ID NO: 58), correspondingto a second common sequence motif in the E6 proteins of HPV strains 16and 45, respectively, or RHLDKKQRFHNIRGRWTGRCMSCC (SEQ ID NO:5) andRHLKDKRRFHSIAGQYRGQCNTCC (SEQ ID NO:59) corresponding to a third commonsequence motif in the E6 proteins of HPV strains 16 and 45,respectively. If a subject antibody binds to other E6 proteins, then itusually binds to the other E6 proteins at positions equivalent to thosediscussed above, or boxed in FIG. 1. For example, the E6 proteins fromHPV58, HPV33, HPV52, HPV31, HPV16, HPV18 and HPV45 are shown in FIG. 2,and the above-referenced motifs are boxed therein.

Accordingly, since antibodies generally recognize motifs smaller thanthose listed above, a subject antibody may recognize peptides that aresmaller than and contained within the motifs described above. Forexample, a subject antibody may bind to a peptide having any 9contiguous amino acids set forth in any one of SEQ NOS: 1, 3, 5, 57, 58and 59. In particular, a subject antibody may recognize the sequencesRPRKLPQLCTEL (SEQ ID NO:7) and RPYKLPDLCTEL (SEQ ID NO:60),corresponding to sub-sequences of the first common sequences of E6proteins of HPV strains 16 and 45, described above, LLIRCINCQKPL (SEQ IDNO:9) and LLIRCLRCQKPL (SEQ ID NO: 61) corresponding to sub-sequences ofthe second common sequences of E6 proteins of HPV strains 16 and 45, asdescribed above, or RHLDKKQRFHNI (SEQ ID NO:11) and RHLKDKRRFHSI (SEQ IDNO: 62) corresponding to sub-sequences of the third common sequences ofE6 proteins of HPV strains 16 and 45, as described above. Since thesesub-sequences are generally conserved between different E6 proteins, asdiscussed above, antibodies that bind to the above-recited sequencesgenerally bind to E6 proteins from other HPV strains. In certainembodiments, cysteine residues can be replaced by serine residues toavoid disulfide bridge formation.

Methods for making antibodies, particular monoclonal antibodies, arewell known in the art and described in various well known laboratorymanuals (e.g., Harlow et al., Antibodies: A Laboratory Manual, FirstEdition (1988) Cold spring Harbor, N.Y.; Harlow and Lane, UsingAntibodies: A Laboratory Manual, CSHL Press (1999) and Ausubel, et al.,Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, (1995)).Accordingly, given the peptide sequences set forth above and in theaccompanying tables, methods for making the subject antibodies do notneed to be described herein in any great detail. Any fragment of alonger full-length E6 protein that contains a subject common motif(e.g., the full length protein), a full length E6 protein, or a fusionprotein thereof may be used to make the subject antibodies. In certainembodiments, a full length E6 protein, a peptide containing a recitedsequence, or a chemically modified (e.g., conjugated) derivative orfusion thereof (e.g., a MBP or GST fusion), may be used as an antigen.In certain embodiments, a nucleic acid encoding the polypeptide may beemployed, or a mixture of different polypeptides (e.g., a mixture of E6polypeptides, each polypeptide from a different HPV strain) may be usedas an antigen (Michel (2002) Vaccine 20:A83-A88). Accordingly an antigenis mixed with an adjuvant, and a suitable non-human animal (e.g., amouse, chicken, goat, rabbit, hamster, horse, rat or guinea pig, etc.)is immunized using standard immunization techniques (e.g., intramuscularinjection) and once a specific immune response of the has beenestablished, blood from the animal may be collected and polyclonalantisera that specifically binds to described peptides may be isolated.In many cases, cells from the spleen of the immunized animal are fusedwith a myeloma cell line, and, after fusion, the cells are grown inselective medium containing e.g., hypoxanthine, aminopterin, andthymidine (HAT), to select for hybridoma growth, and after 2-3 weeks,hybridoma colonies appear. Supernatants from these cultured hybridomacells are screened for antibody secretion, usually by enzyme-linkedimmunosorbent assay (ELISA) or the like, and positive clones secretingmonoclonal antibodies specific for the antigen can be selected andexpanded according to standard procedures.

Exemplary peptides suitable for immunizations are described in Table 1.The peptides are shown as a “consensus” sequence (i.e. peptides in whichone of several amino acids may exist at one or more positions) in orderto indicate that any one or a mixture of different peptides that aredescribed by the consensus could be used to make the subject antibodies.Accordingly, when a consensus sequence is described, every individualpeptide that falls within the consensus should be considered explicitlydescribed. In particular embodiments, exemplary species of peptideencompassed by the consensus sequences have a sequence found in anaturally-occurring HPV E6 protein, such as those described in FIG. 1.Such exemplary sequences can be identified as sequences starting at theamino acid positions defined by the third column of Table 1, “StartingAA” of particular HPV types “HPV type”, and corresponding positions ofother HPV E6 proteins (i.e., those positions that are aligned with thepositions indicated in Table 1).

Accordingly, peptides having 9, 10, 11, 12, 13, 14, 15 or more, usuallyup to about 20 or more contiguous amino acids of any of the peptidesdescribed above may be used for immunizations. In some embodiments, arecited peptide sequence may be contained within a larger peptide thatmay be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more, sometimes up to about 15or 20 or more amino acids greater in size than a recited polypeptide.Accordingly, a subject peptide may be from about 8 to about 30 aminoacids in length. In certain embodiments, a subject peptide is about 9-20amino acids in length, and usually contains an amino acid sequencedescribed above.

Accordingly, depending on the antibodies desired, a suitable animal isimmunized with a subject peptide or a mixture of subject peptides (e.g.,a mixture of 2, 3, 4, 5 about 6 or more, about 10 or more or about 15 ormore, usually up to about 20 or 30 or more peptides described above).Antibodies are usually isolated from the animal and tested for bindingto different HPV E6 proteins using standard methods (e.g., ELISA,western blot, etc.). In many embodiments, therefore, antibodies will bescreened for binding to E6 proteins from HPV strains 16 and 18, HPVstrains 16, 18, 31, 33 and 45, or, in certain embodiments, all of theHPV strains shown in FIG. 1 or 2, and maybe others. Accordingly,antibodies that bind to, i.e., cross-react with, E6 proteins from morethan one strain of HPV may be identified, and permanent cell linesproducing those antibodies may be established using known methods. Inother words, antibodies are usually tested for binding to more than oneantigen, and those antigens are usually E6 proteins from various HPVstrains, or fragments thereof. In most embodiments, the antibodies willbe tested for binding to antigens in native and denatured states.Antibodies that bind to a plurality of E6 proteins have desirablebinding properties, and, accordingly, find use in the subject methods.

As is well known in the art, the subject antibodies may be conjugated toa detectable label, or may be part of a signal generating system, asdescribed above.

Accordingly, using the methods set forth above, an antibody compositionfor detecting a plurality of HPV E6 proteins is provided. In certainembodiments, a mixture of different antibodies that recognize at least5, 7, 9, 12, 15, 20 or 24 different strains of HPV may be employed. Thecomposition may contain at least one antibody that recognizes at least 3different oncogenic E6 proteins. The composition may contain 1, 2, 3, 4,or 5 or more different antibodies, each antibody of the compositionrecognizing at least one (e.g., 2, 3, about 5, about 10, etc.) E6protein. Collectively, the antibodies bind to all or a portion of the E6proteins shown in FIG. 1, and, in certain embodiments, may also bind tonon-oncogenic E6 proteins. The antibodies may be mixed, or separate fromeach other, i.e., in different vessels.

Any of the above-described antibodies may bind to an epitope set forthin Table 1.

TABLE 1 Epitopes HPV Starting Sequence type AA(K/R)-(K/R)-R-F-H-(N/K/S/E/R)-I-(A/S) 59 124F-H-(N/K/S/E/R)-I-(A/S)-(G/H)-X-(W/Y) 59 127H-(N/K/S/E/R)-I-(A/S)-(G/H)-(R/Q)-(W/Y)-(T/K/R) 59 128P-(E/A/Q)-E-K-(Q/L/K/R)-(R/K/L)-(H/V/I/L)-(V/L/C) 26 112(G/H)-(R/Q/T/M/G/A/Y/H/S/N/I)-(W/Y/F)- 59 132(T/R/K/A)-G-(R/Q/S/L)-C-(R/L/M/A/T)(W/Y/F)-(T/R/K/A)-G-(R/Q/S/L)-C-(R/L/M/A/T)- 59 134(L/R/A/T)-(N/R/S/A/Q/G)G-(R/Q/S/L)-C-(R/L/M/A/T)-(L/R/A/T)-(N/R/S/A/Q/G)- 59 136 C-(W/C/R)(R/K)-P-(R/Y)-(K/T/S)-(L/V)-(H/P)-(D/E/H/Q)-L 59 10(M/R/L)-F-(E/Q/D/H)-(D/N)-(P/T)-(Q/R/A/E/T)- 59 3 (E/Q)-(R/K)(D/N)-(P/T)-(Q/R/A/E/T)-(E/Q)-(R/K)-(R/K)-P- 59 6 (R/Y)(L/V)-(H/P)-(D/E/Q)-L-(C/S)-(E/T/Q)-(E/V/A/T)- 59 14 (L/V)-(N/E/D)(D/E/N)-(L/V/I)-(Q/E/R/T)-(L/V/I)-(Q/N/D/S/A/N)- 59 26 C-V-(F/Y/E)-L-(L/S)-I-R-C-(I/Y/H/L/M)-(R/I/C)-C 59 101(R/I/C)-C-(Q/L)-(K/R)-P-L-(C/T/G/N)-P 59 107(K/R)-P-L-(C/T/G/N)-P-(E/A/Q)-E-K 59 110P-(E/A/Q)-E-K-(Q/L/K)-(R/L/K)-(H/I)-(L/V/C) 26 112K-(Q/L/K)-(R/L/K)-(H/I)-(L/V/C)-(D/E/N)- 26 115 (E/D/Y/L/K/S)-(K/N)(L/V/C)-(D/E/N)-(E/D/Y/L/K/S)-(K/N)-(K/R)-R- 26 119 F-HI-(A/S)-(G/H)-(R/Q)-(W/Y)-(T/K/R)-G-(R/Q/L/S) 26 128(W/Y)-(T/K/R)-G-(R/Q/L/S)-C-(M/A/L/R/T)- 26 132 (N/S/A/R)-C

Certain hybridomas that produce the monoclonal antibodies describedabove and below may be deposited at the ATCC. Any of the depositedhybridomas, the antibodies produced by those hybridomas, as well asother antibodies that bind the same epitopes as the antibodies producedby those hybridomas, are also embodiments of this invention and may beclaimed herein. Such antibodies may be employed in any of the methodsdescribed herein.

Methods for Detecting an HPV E6 Protein in a Sample

The invention provides a method of detecting an HPV E6 protein in asample. In general, the methods involve contacting a subject antibodycomposition with a sample, and assessing any binding of the antibody tothe sample. In most embodiments, binding of the antibody to the sampleindicates the presence of an HPV E6 protein.

The antibodies of the invention may be screened for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, and cellular immunostaining (fixedor native) assays to name but a few. Such assays are routine and wellknown in the art (see, e.g., Ausubel et al., eds, 1994, CurrentProtocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., NewYork, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

Immunoprecipitation protocols generally involve lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein sepharose beads to the cell lysate, incubating for about an houror more at 4° C., washing the beads in lysis buffer and resuspending thebeads in SDS/sample buffer. The ability of the antibody of interest toimmunoprecipitate a particular antigen can be assessed by, e.g., westernblot analysis. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the binding of the antibodyto an antigen and decrease the background (e.g., pre-clearing the celllysate with sepharose beads).

Western blot analysis generally involves preparation of protein samplesfollowed by electrophoresis of the protein samples in a polyacrylamidegel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of theantigen), and transfer of the separated protein samples from thepolyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon.Following transfer, the membrane is blocked in blocking solution (e.g.,PBS with 3% BSA or non-fat milk), washed in washing buffer (e.g.,PBS-Tween 20), and incubated with primary antibody (the antibody ofinterest) diluted in blocking buffer. After this incubation, themembrane is washed in washing buffer, incubated with a secondaryantibody (which recognizes the primary antibody, e.g., an anti-humanantibody) conjugated to an enzymatic substrate (e.g., horseradishperoxidase or alkaline phosphatase) or radioactive molecule (e.g., 32Por 125I), and after a further wash, the presence of the antigen may bedetected. One of skill in the art would be knowledgeable as to theparameters that can be modified to increase the signal detected and toreduce the background noise.

ELISAs involve preparing antigen, coating the well of a 96 wellmultiwell plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., ³H or ¹²⁵I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest conjugated to a labeled compound (e.g., ³H or ¹²⁵I)in the presence of increasing amounts of an unlabeled second antibody.

Antibodies of the invention may be screened using immunocytochemistymethods on cells (e.g., mammalian cells, such as CHO cells) transfectedwith a vector enabling the expression of an antigen or with vector aloneusing techniques commonly known in the art. Antibodies that bind antigentransfected cells, but not vector-only transfected cells, are antigenspecific.

In certain embodiments, however, the assay is an antigen capture assay,and an array or microarray of antibodies may be employed for thispurpose. Methods for making and using microarrays of polypeptides areknown in the art (see e.g. U.S. Pat. Nos. 6,372,483, 6,352,842,6,346,416 and 6,242,266).

Systems for Detecting an Oncogenic HPV E6 Protein

The invention provides a system for detecting the presence of anoncogenic HPV E6 polypeptide in a sample. In general, the systemcomprises a first and a second binding partner for an oncogenic HPV E6polypeptide. In most embodiments, the first binding partner is a PDZdomain protein and the second binding partner is a subject antibody.

The subject antibodies may be used along with certain PDZ domainproteins as part of a system for detecting E6 protein from oncogenicstrains of HPV. As mentioned above, oncogenic HPV E6 proteins contain a“PDZ-ligand” (“PL”) that is bound by certain PDZ domain polypeptides.Non-oncogenic HPV E6 proteins do not contain such a PDZ-ligand, and,accordingly, are not bound by PDZ-domain polypeptides. Many PDZ domainssuitable for use in the subject system are generally described in Table2, and include MAGI-1 PDZ domain 2, the PDZ domain of TIP-1, and the PDZdomains 1 and 2 of DLG1, and many others. As would be recognized by oneof skill in the art, a PDZ domain may be employed as part of a fusionprotein, particularly in embodiments in which the PDZ domain polypeptideis anchored to a substrate. Accordingly, the subject system generallycontains a suitable PDZ domain polypeptide, which is usually a fusionprotein, and a subject antibody.

In certain embodiments, one of the binding partners is attached to asolid support, and the other binding partner may be labeled or part of asignal producing system. Proteins may be covalently bound ornoncovalently attached through nonspecific bonding. If covalent bondingbetween the fusion protein and the surface is desired, the surface willusually be polyfunctional or be capable of being polyfunctionalized.Functional groups which may be present on the surface and used forlinking can include carboxylic acids, aldehydes, amino groups, cyanogroups, ethylenic groups, hydroxyl groups, mercapto groups and the like.The manner of linking a wide variety of compounds to various surfaces iswell known and is amply illustrated in the literature.

TABLE 2 SEQ ID GI or NO. name Acc. # Sequence 106 AF6 domain 1 430993LRKEPEIITVTLKKQNGMGLSIVAAKGAGQDKLGIYVKSVVKGGAADVDGRLAAGDQLLSVDGRSLVGLSQERAAEL MTRTSSVVTLEVAKQG 107 AIPC domain1 12751451 LIRPSVISIIGLYKEKGKGLGFSIAGGRDCIRGQMGIFVKTIFPNGSAAEDGRLKEGDEILDVNGIPIKGLTFQEAIHTFKQI RSGLFVLTVRTKLVSPSLTNSS 108AIPC domain 3 12751451 QSENEEDVCFIVLNRKEGSGLGFSVAGGTDVEPKSITVHRVFSQGAASQEGTMNRGDFLLSVNGASLAGLAHGNVLKV LHQAQLHKDALVVIKKGMDQPRPSNSS 109AIPC domain 2 12751451 GISSLGRKTPGPKDRIVMEVTLNKEPRVGLGIGACCLALENSPPGIYIHSLAPGSVAKMESNLSRGDQILEVNSVNVRHAALSKVHAILSKCPPGPVRLVIGRHPNPKVSEQEMDEVIAR STYQESKEANSS 110 AIPC domain 412751451 LGRSVAVHDALCVEVLKTSAGLGLSLDGGKSSVTGDGPLVIKRVYKGGAAEQAGIIEAGDEILAINGKPLVGLMHFD AWNIMKSVPEGPVQLLIRKHRNSS 111 ALPdomain 1 2773059 REEGGMPQTVILPGPAPWGFRLSGGIDFNQPLVITRITPGSKAAAANLCPGDVILAIDGFGTESMTHADAQDRIKAAAH QLCLKIDRGETHLWSPNSS 112 APXL1domain 1 13651263 ILVEVQLSGGAPWGFTLKGGREHGEPLVITKIEEGSKAAAVDKLLAGDEIVGINDIGLSGFRQEAICLVKGSHKTLKLVV KRNSS 113 CARD11 domain 112382772 SVGHVRGPGPSVQHTTLNGDSLTSQLTLLGGNARGSFVHSVKPGSLAEKAGLREGHQLLLLEGCIRGERQSVPLDTCT KEEAHWTIQRCSGPVTLHYKVNHEGYRK 114CARD14 domain 1 13129123 RRPARRILSQVTMLAFQGDALLEQISVIGGNLTGIFIHRVTPGSAADQMALRPGTQIVMVDYEASEPLFKAVLEDTTLEE AVGLLRRVDGFCCLSVKVNTDGYKR 115CARD14 domain 1 13129123 ILSQVTMLAFQGDALLEQISVIGGNLTGIFIHRVTPGSAADQMALRPGTQIVMVDYEASEPLFKAVLEDTTLEEAVGLLR RVDGFCCLSVKVNTDGYKRL 116 CASKdomain 1 3087815 TRVRLVQFQKNTDEPMGITLKMNELNHCIVARIMHGGMIHRQGTLHVGDEIREINGISVANQTVEQLQKMLREMRGSI TFKIVPSYRTQS 117 CNK1 domain 13930780 LEQKAVLEQVQLDSPLGLEIHTTSNCQHFVSQVDTQVPTDSRLQIQPGDEVVQINEQVVVGWPRKNMVRELLREPAG LSLVLKKIPIP 118 Cytohesin binding3192908 QRKLVTVEKQDNETFGFEIQSYRPQNQNACSSEMFTLICK Protein domain 1IQEDSPAHCAGLQAGDVLANINGVSTEGFTYKQVVDLIR SSGNLLTIETLNG 119 Densin domain1 16755892 RCLIQTKGQRSMDGYPEQFCVRIEKNPGLGFSISGGISGQGNPFKPSDKGIFVTRVQPDGPASNLLQPGDKILQANGHSF VHMEHEKAVLLLKSFQNTVDLVIQRELTV120 DLG 6 splice AB053303 PTSPEIQELRQMLQAPHFKGATIKRHEMTGDILVARIIHGvariant 2 domain 1 GLAERSGLLYAGDKLVEVNGVSVEGLDPEQVIHILAMSRGTIMFKVVPVSDPPVNSS 121 DLG 6 splice 14647140PTSPEIQELRQMLQAPHFKALLSAHDTIAQKDFEPLLPPLP variant 1 domain 1DNIPESEEAMRIVCLVKNQQPLGATIKRHEMTGDILVARIIHGGLAERSGLLYAGDKLVEVNGVSVEGLDPEQVIHILAM SRGTIMFKVVPVSDPPVNSS 122 DLG1domain 1 475816 IQVNGTDADYEYEEITLERGNSGLGFSIAGGTDNPHIGDDSSIFITKIITGGAAAQDGRLRVNDCILQVNEVDVRDVTHS KAVEALKEAGSIVRLYVKRRN 123 DLG1domain 2 475816 IQLIKGPKGLGFSIAGGVGNQHIPGDNSIYVTKIIEGGAAHKDGKLQIGDKLLAVNNVCLEEVTHEEAVTALKNTSDFV YLKVAKPTSMYMNDGN 124 DLG1 domains1 475816 VNGTDADYEYEEITLERGNSGLGFSIAGGTDNPHIGDDSS and 2IFITKIITGGAAAQDGRLRVNDCILQVNEVDVRDVTHSKAVEALKEAGSIVRLYVKRRKPVSEKIMEIKLIKGPKGLGFSIAGGVGNQHIPGDNSIYVTKIIEGGAAHKDGKLQIGDKLLAVNNVCLEEVTHEEAVTALKNTSDFVYLKVAKPTSMYM NDGYA 125 DLG1 domain 3 475816ILHRGSTGLGFNIVGGEDGEGIFISFILAGGPADLSGELRKGDRIISVNSVDLRAASHEQAAAALKNAGQAVTIVAQYRP EEYSR 126 DLG2 domain 3 12736552IEGRGILEGEPRKVVLHKGSTGLGFNIVGGEDGEGIFVSFILAGGPADLSGELQRGDQILSVNGIDLRGASHEQAAAALK GAGQTVTIIAQHQPEDYARFEAKIHDLNSS127 DLG2 domain 1 12736552 ISYVNGTEIEYEFEEITLERGNSGLGFSIAGGTDNPHIGDDPGIFITKIIPGGAAAEDGRLRVNDCILRVNEVDVSEVSHSK AVEALKEAGSIVRLYVRRR 128 DLG2domain 2 12736552 IPILETVVEIKLFKGPKGLGFSIAGGVGNQHIPGDNSIYVTKIIDGGAAQKDGRLQVGDRLLMVNNYSLEEVTHEEAVAILKNTSEVVYLKVGKPTTIYMTDPYGPPNSSLTD 129 DLG5 domain 1 3650451GIPYVEEPRHVKVQKGSEPLGISIVSGEKGGIYVSKVTVGSIAHQAGLEYGDQLLEFNGINLRSATEQQARLIIGQQCDTI TILAQYNPHVHQLRNSSLTD 130 DLG5domain 2 3650451 GILAGDANKKTLEPRVVFIKKSQLELGVHLCGGNLHGVFVAEVEDDSPAKGPDGLVPGDLILEYGSLDVRNKTVEEVY VEMLKPRDGVRLKVQYRPEEFIVTD 131DVL1 domain 1 2291005 LNIVTVTLNMERHHFLGISIVGQSNDRGDGGIYIGSIMKGGAVAADGRIEPGDMLLQVNDVNFENMSNDDAVRVLREI VSQTGPISLTVAKCW 132 DVL2 domain 12291007 LNIITVTLNMEKYNFLGISIVGQSNERGDGGIYIGSIMKGGAVAADGRIEPGDMLLQVNDMNFENMSNDDAVRVLRDI VHKPGPIVLTVAKCWDPSPQNS 133 DVL3domain 1 6806886 IITVTLNMEKYNFLGISIVGQSNERGDGGIYIGSIMKGGAVAADGRIEPGDMLLQVNEINFENMSNDDAVRVLREIVHKP GPITLTVAKCWDPSP 134 EBP50 domain2 3220018 QQRELRPRLCTMKKGPSGYGFNLHSDKSKPGQFIRSVDPDSPAEASGLRAQDRIVEVNGVCMEGKQHGDVVSAIRAG GDETKLLVVDRETDEFFKNSS 135 EBP50domain 1 3220018 GIQMSADAAAGAPLPRLCCLEKGPNGYGFHLHGEKGKLGQYIRLVEPGSPAEKAGLLAGDRLVEVNGENVEKETHQQVVSRIRAALNAVRLLVVDPETDEQLQKLGVQVREELLRAQEAPGQAEPPAAAEVQGAGNENEPREADKSHPEQREL RN 136 EBP50 domains 1 3220018GIQMSADAAAGAPLPRLCCLEKGPNGYGFHLHGEKGKL and 2GQYIRLVEPGSPAEKAGLLAGDRLVEVNGENVEKETHQQVVSRIRAALNAVRLLVVDPETDEQLQKLGVQVREELLRAQEAPGQAEPPAAAEVQGAGNENEPREADKSHPEQRELRPRLCTMKKGPSGYGFNLHSDKSKPGQFIRSVDPDSPAEASGLRAQDRIVEVNGVCMEGKQHGDVVSAIRAGGDETK LLVVDRETDEFFK 137 EBP50 domain 13220018 QMSADAAAGAPLPRLCCLEKGPNGYGFHLHGEKGKLGQYIRLVEPGSPAEKAGLLAGDRLVEVNGENVEKETHQQVVSRIRAALNAVRLLVVDPETDEQLQKLGVQVREELLRAQEAPGQAEPPAAAEVQGAGNENEPREADKSHPEQRELRNSS 138 ELFIN 1 domain 1 2957144LTTQQIDLQGPGPWGFRLVGGKDFEQPLAISRVTPGSKAALANLCIGDVITAIDGENTSNMTHLEAQNRIKGCTDNLTL TVARSEHKVWSPLVTNSSW 139 ENIGMAdomain 1 561636 IFMDSFKVVLEGPAPWGFRLQGGKDFNVPLSISRLTPGGKAAQAGVAVGDWVLSIDGENAGSLTHIEAQNKIRACGER LSLGLSRAQPV 140 ERBIN domain 18923908 QGHELAKQEIRVRVEKDPELGFSISGGVGGRGNPFRPDDDGIFVTRVQPEGPASKLLQPGDKIIQANGYSFINIEHGQAV SLLKTFQNTVELIIVREVSS 141FLJ00011 domain 1 10440352 KNPSGELKTVTLSKMKQSLGISISGGIESKVQPMVKIEKIFPGGAAFLSGALQAGFELVAVDGENLEQVTHQRAVDTIR RAYRNKAREPMELVVRVPGPSPRPSPSD 142FLJ11215 domain 1 11436365 EGHSHPRVVELPKTEEGLGFNIMGGKEQNSPIYISRIIPGGIADRHGGLKRGDQLLSVNGVSVEGEHHEKAVELLKAAQ GKVKLVVRYTPKVLEEME 143 FLJ12428domain 1 BC012040 PGAPYARKTFTIVGDAVGWGFVVRGSKPCHIQAVDPSGPAAAAGMKVCQFVVSVNGLNVLHVDYRTVSNLILTGPRT IVMEVMEELEC 144 FLJ12615 domain 110434209 GQYGGETVKIVRIEKARDIPLGATVRNEMDSVIISRIVKGGAAEKSGLLHEGDEVLEINGIEIRGKDVNEVFDLLSDMH GTLTFVLIPSQQIKPPPA 145 FLJ21687domain 1 10437836 KPSQASGHFSVELVRGYAGFGLTLGGGRDVAGDTPLAVRGLLKDGPAQRCGRLEVGDLVLHINGESTQGLTHAQAV ERIRAGGPQLHLVIRRPLETHPGKPRGV 146FLJ31349 domain 1 AK055911 PVMSQCACLEEVHLPNIKPGEGLGMYIKSTYDGLHVITGTTENSPADRSQKIHAGDEVTQVNQQTVVGWQLKNLVKK LRENPTGVVLLLKKRPTGSFNFTP 147FLJ32798 domain 1 AK057360 IDDEEDSVKIIRLVKNREPLGATIKKDEQTGAIIVARIMRGGAADRSGLIHVGDELREVNGIPVEDKRPEEIIQILAQSQG AITFKIIPGSKEETPS 148 GORASP 213994253 MGSSQSVEIPGGGTEGYHVLRVQENSPGHRAGLEPFFDFI domains 1 and 2VSINGSRLNKDNDTLKDLLKANVEKPVKMLIYSSKTLELRETSVTPSNLWGGQGLLGVSIRFCSFDGANENVWHVLEVESNSPAALAGLRPHSDYIIGADTVMNESEDLFSLIETHEAKPLKLYVYNTDTDNCREVIITPNSAWGGEGSLGCGIGYGYLHRIPTRPFEEGKKISLPGQMAGTPITPLKDGFTEVQLSSVNPPSLSPPGTTGIEQSLTGLSISSTPPAVSSVLSTGVPTVPLLPPQVNQSLTSVPPMNPATTLPGLMPLPAGLPNLPNLNLNLPAPHIMPGVGLPELVNPGLPPLPSMPPRNLPGIAPLPLPSEFLPSFPLVPESSSAASSGELLSSLPPTSNAPSDPATTTAKADAASSLTVDVTPPTAKAPTTVEDRVGDSTPVSEKPVSA AVDANASESP 149 GORASP 2 domain 213994253 NENVWHVLEVESNSPAALAGLRPHSDYIIGADTVMNESEDLFSLIETHEAKPLKLYVYNTDTDNCREVIITPNSAWGGE GSLGCGIGYGYLHRIPTR 150 GORASP 2domain 1 13994253 MGSSQSVEIPGGGTEGYHVLRVQENSPGHRAGLEPFFDFIVSINGSRLNKDNDTLKDLLKANVEKPVKMLIYSSKTLEL RETSVTPSNLWGGQGLLGVSIRFCSFDGANE151 GORASP 1 domain 2 29826292 RASEQVWHVLDVEPSSPAALAGLRPYTDYVVGSDQILQESEDFFTLIESHEGKPLKLMVYNSKSDSCREVTVTPNAAW GGEGSLGCGIGYGYLHRIPTQ 152 GORASP1 domain 1 29826292 MGLGVSAEQPAGGAEGFHLHGVQENSPAQQAGLEPYFDFIITIGHSRLNKENDTLKALLKANVEKPVKLEVFNMKTMRVREVEVVPSNMWGGQGLLGASVRFCSFRRASE 153 GORASP 1 29826292MGLGVSAEQPAGGAEGFHLHGVQENSPAQQAGLEPYFD domains 1 and 2FIITIGHSRLNKENDTLKALLKANVEKPVKLEVFNMKTMRVREVEVVPSNMWGGQGLLGASVRFCSFRRASEQVWHVLDVEPSSPAALAGLRPYTDYVVGSDQILQESEDFFTLIESHEGKPLKLMVYNSKSDSCREVTVTPNAAWGGEGSLGCGIGYGYLHRIPTQPPSYHKKPPGTPPPSALPLGAPPPDALPPGPTPEDSPSLETGSRQSDYMEALLQAPGSSMEDPLPGPGSPSHSAPDPDGLPHFMETPLQPPPPVQRVMDPGFLDVSGISLLDNSNASVWPSLPSSTELTTTAVSTSGPEDICSSSSSHERGGEATWSGSEFEVSFLDSPGAQAQADHLPQLTLPDSLTSAASPEDGLSAELLEAQAEEEPASTEGLDTGTEAEGLDSQ AQISTTE 154 GRIP 1 domain 64539083 IYTVELKRYGGPLGITISGTEEPFDPIIISSLTKGGLAERTGAIHIGDRILAINSSSLKGKPLSEAIHLLQMAGETVTLKIKK QTDAQSA 155 GRIP 1 domain 14539083 VVELMKKEGTTLGLTVSGGIDKDGKPRVSNLRQGGIAARSDQLDVGDYIKAVNGINLAKFRHDEIISLLKNVGERVVLE VEYE 156 GRIP 1 domain 34539083 HVATASGPLLVEVAKTPGASLGVALTTSMCCNKQVIVIDKIKSASIADRCGALHVGDHILSIDGTSMEYCTLAEATQFL ANTTDQVKLEILPHHQTRLALKGPNSS 157GRIP 1 domain 7 4539083 IMSPTPVELHKVTLYKDSDMEDFGFSVADGLLEKGVYVKNIRPAGPGDLGGLKPYDRLLQVNHVRTRDFDCCLVVPL IAESGNKLDLVISRNPLA 158 GRIP 1domain 4 4539083 IYTVELKRYGGPLGITISGTEEPFDPIIISSLTKGGLAERTGAIHIGDRILAINSSSLKGKPLSEAIHLLQMAGETVTLKIKK QTDAQSA 159 GRIP 1 domain 54539083 IMSPTPVELHKVTLYKDSDMEDFGFSVADGLLEKGVYVKNIRPAGPGDLGGLKPYDRLLQVNHVRTRDFDCCLVVPL IAESGNKLDLVISRNPLA 160 GTPaseactivating 2389008 SRGCETRELALPRDGQGRLGFEVDAEGFVTHVERFTFAE enzyme domain1 TAGLRPGARLLRVCGQTLPSLRPEAAAQLLRSAPKVCVT VLPPDESGRP 161 Guanineexchange 6650765 CSVMIFEVVEQAGAIILEDGQELDSWYVILNGTVEISHPD factor domain1 GKVENLFMGNSFGITPTLDKQYMHGIVRTKVDDCQFVCI AQQDYWRILNHVEKNTHKVEEEGEIVMVH162 HEMBA 1000505 10436367 PRETVKIPDSADGLGFQIRGFGPSVVHAVGRGTVAAAAGdomain 2 LHPGQCIIKVNGINVSKETHASVIAHVTACRKYRRPTKQD SIQ 163 HEMBA 100050510436367 LENVIAKSLLIKSNEGSYGFGLEDKNKVPIIKLVEKGSNA domain 1EMAGMEVGKKIFAINGDLVFMRPFNEVDCFLKSCLNSRK PLRVLVSTKP 164 HEMBA 10031177022001 EDFCYVFTVELERGPSGLGMGLIDGMHTHLGAPGLYIQT domain 1LLPGSPAAADGRLSLGDRILEVNGSSLLGLGYLRAVDLIR HGGKKMRFLVAKSDVETAKKI 165hShroom domain 1 18652858 IYLEAFLEGGAPWGFTLKGGLEHGEPLIISKVEEGGKADTLSSKLQAGDEVVHINEVTLSSSRKEAVSLVKGSYKTLRL VVRRDVCTDPGH 166 HSPC227 domain1 7106843 NNELTQFLPRTITLKKPPGAQLGFNIRGGKASQLGIFISKVIPDSDAHRAGLQEGDQVLAVNDVDFQDIEHSKAVEILKT AREISMRVRFFPYNYHRQKE 167 HTRA 3domain 1 AY040094 FLTEFQDKQIKDWKKRFIGIRMRTITPSLVDELKASNPDFPEVSSGIYVQEVAPNSPSQRGGIQDGDIIVKVNGRPLVDSS ELQEAVLTESPLLLEVRRGNDDLLFS 168HTRA 4 domain 1 AL576444 NKKYLGLQMLSLTVPLSEELKMHYPDFPDVSSGVYVCKVVEGTAAQSSGLRDHDVIVNINGKPITTTTDVVKALDSD SLSMAVLRGKDNLLLTV 169 INADLdomain 3 2370148 PGSDSSLFETYNVELVRKDGQSLGIRIVGYVGTSHTGEASGIYVKSIIPGSAAYHNGHIQVNDKIVAVDGVNIQGFANHD VVEVLRNAGQVVHLTLVRRKTSSSTSRIHRD170 INADL domain 8 2370148 PATCPIVPGQEMIIEISKGRSGLGLSIVGGKDTPLNAIVIHEVYEEGAAARDGRLWAGDQILEVNGVDLRNSSHEEAITA LRQTPQKVRLVVY 171 INADL domain 22370148 LPETVCWGHVEEVELINDGSGLGFGIVGGKTSGVVVRTIVPGGLADRDGRLQTGDHILKIGGTNVQGMTSEQVAQVL RNCGNSVRMLVARDPAGDIQSPI 172 INADLdomain 6 2370148 PNFSHWGPPRIVEIFREPNVSLGISIVVGQTVIKRLKNGEELKGIFIKQVLEDSPAGKTNALKTGDKILEVSGVDLQNASHSEAVEAIKNAGNPVVFIVQSLSSTPRVIPNVHNKANSS 173 INADL domain 7 2370148PGELHIIELEKDKNGLGLSLAGNKDRSRMSIFVVGINPEGPAAADGRMRIGDELLEINNQILYGRSHQNASAIIKTAPSK VKLVFIRNEDAVNQMANSS 174 INADLdomain 5 2370148 LSSPEVKIVELVKDCKGLGFSILDYQDPLDPTRSVIVIRSLVADGVAERSGGLLPGDRLVSVNEYCLDNTSLAEAVEILK AVPPGLVHLGICKPLVEFIVTD 175 INADLdomain 1 2370148 IWQIEYIDIERPSTGGLGFSVVALRSQNLGKVDIFVKDVQPGSVADRDQRLKENDQILAINHTPLDQNISHQQAIALLQQ TTGSLRLIVAREPVHTKSSTSSSE 176INADL domain 4 2370148 NSDDAELQKYSKLLPIHTLRLGVEVDSFDGHHYISSIVSGGPVDTLGLLQPEDELLEVNGMQLYGKSRREAVSFLKEVP PPFTLVCCRRLFDDEAS 177 KIAA0313domain 1 7657260 HLRLLNIACAAKAKRRLMTLTKPSREAPLPFILLGGSEKGFGIFVDSVDSGSKATEAGLKRGDQILEVNGQNFENIQLSKAMEILRNNTHLSITVKTNLFVFKELLTRLSEEKRNGAP 178 KIAA0316 domain 1 6683123IPPAPRKVEMRRDPVLGFGFVAGSEKPVVVRSVTPGGPSEGKLIPGDQIVMINDEPVSAAPRERVIDLVRSCKESILLTVI QPYPSPK 179 KIAA0340 domain 12224620 LNKRTTMPKDSGALLGLKVVGGKMTDLGRLGAFITKVKKGSLADVVGHLRAGDEVLEWNGKPLPGATNEEVYNIILE SKSEPQVEIIVSRPIGDIPRIHRD 180KIAA0380 domain 1 2224700 RCVIIQKDQHGFGFTVSGDRIVLVQSVRPGGAAMKAGVKEGDRIIKVNGTMVTNSSHLEVVKLIKSGAYVALTLLGS 181 KIAA0382 domain 1 7662087ILVQRCVIIQKDDNGFGLTVSGDNPVFVQSVKEDGAAMRAGVQTGDRIIKVNGTLVTHSNHLEVVKLIKSGSYVALTV QGRPPGNSS 182 KIAA0440 domain 12662160 SVEMTLRRNGLGQLGFHVNYEGIVADVEPYGYAWQAGLRQGSRLVEICKVAVATLSHEQMIDLLRTSVTVKVVIIPPH 183 KIAA0545 domain 1 14762850LKVMTSGWETVDMTLRRNGLGQLGFHVKYDGTVAEVEDYGFAWQAGLRQGSRLVEICKVAVVTLTHDQMIDLLRT SVTVKVVIIPPFEDGTPRRGW 184KIAA0559 domain 1 3043641 HYIFPHARIKITRDSKDHTVSGNGLGIRIVGGKEIPGHSGEIGAYIAKILPGGSAEQTGKLMEGMQVLEWNGIPLTSKTYE EVQSIISQQSGEAEICVRLDLNML 185KIAA0613 domain 1 3327039 SYSVTLTGPGPWGFRLQGGKDFNMPLTISRITPGSKAAQSQLSQGDLVVAIDGVNTDTMTHLEAQNKIKSASYNLSLTL QKSKNSS 186 KIAA0858 domain 14240204 FSDMRISINQTPGKSLDFGFTIKWDIPGIFVASVEAGSPAEFSQLQVDDEIIAINNTKFSYNDSKEWEEAMAKAQETGHL VMDVRRYGKAGSPE 187 KIAA0902domain 1 4240292 QSAHLEVIQLANIKPSEGLGMYIKSTYDGLHVITGTTENSPADRCKKIHAGDEVIQVNHQTVVGWQLKNLVNALREDP SGVILTLKKRPQSMLTSAPA 188 KIAA0967domain 1 4589577 ILTQTLIPVRHTVKIDKDTLLQDYGFHISESLPLTVVAVTAGGSAHGKLFPGDQILQMNNEPAEDLSWERAVDILREAED SLSITVVRCTSGVPKSSNSS 189KIAA1202 domain 1 6330421 RSFQYVPVQLQGGAPWGFTLKGGLEHCEPLTVSKIEDGGKAALSQKMRTGDELVNINGTPLYGSRQEALILIKGSFRIL KLIVRRRNAPVS 190 KIAA1222domain 1 6330610 ILEKLELFPVELEKDEDGLGISIIGMGVGADAGLEKLGIFVKTVTEGGAAQRDGRIQVNDQIVEVDGISLVGVTQNFAAT VLRNTKGNVRFVIGREKPGQVSE 191KIAA1284 domain 1 6331369 KDVNVYVNPKKLTVIKAKEQLKLLEVLVGIIHQTKWSWRRTGKQGDGERLVVHGLLPGGSAMKSGQVLIGDVLVAVNDVDVTTENIERVLSCIPGPMQVKLTFENAYDVKRET 192 KIAA1389 domain 1 7243158TRGCETVEMTLRRNGLGQLGFHVNFEGIVADVEPFGFAWKAGLRQGSRLVEICKVAVATLTHEQMIDLLRTSVTVK VVIIQPHDDGSPRR 193 KIAA1415domain 1 7243210 VENILAKRLLILPQEEDYGFDIEEKNKAVVVKSVQRGSLAEVAGLQVGRKIYSINEDLVFLRPFSEVESILNQSFCSRRPL RLLVATKAKEIIKIP 194 KIAA1526domain 1 5817166 PDSAGPGEVRLVSLRRAKAHEGLGFSIRGGSEHGVGIYVSLVEPGSLAEKEGLRVGDQILRVNDKSLARVTHAEAVKAL KGSKKLVLSVYSAGRIPGGYVTNH 195KIAA1526 domain 2 5817166 LQGGDEKKVNLVLGDGRSLGLTIRGGAEYGLGIYITGVDPGSEAEGSGLKVGDQILEVNGRSFLNILHDEAVRLLKSSR HLILTVKDVGRLPHARTTVDE 196KIAA1620 domain 1 10047316 LRRAELVEIIVETEAQTGVSGINVAGGGKEGIFVRELREDSPAARSLSLQEGDQLLSARVFFENFKYEDALRLLQCAEP YKVSFCLKRTVPTGDLALR 197 KIAA1719domain 5 1267982 IQTTGAVSYTVELKRYGGPLGITISGTEEPFDPIVISGLTKRGLAERTGAIHVGDRILAINNVSLKGRPLSEAIHLLQVAGE TVTLKIKKQLDR 198 KIAA1719domain 6 1267982 ILEMEELLLPTPLEMHKVTLHKDPMRHDFGFSVSDGLLEKGVYVHTVRPDGPAHRGGLQPFDRVLQVNHVRTRDFDC CLAVPLLAEAGDVLELIISRKPHTAHSS 199KIAA1719 domain 2 1267982 IHTVANASGPLMVEIVKTPGSALGISLTTTSLRNKSVITIDRIKPASVVDRSGALHPGDHILSIDGTSMEHCSLLEATKLL ASISEKVRLEILPVPQSQRPL 200KIAA1719 domain 1 1267982 ITVVELIKKEGSTLGLTISGGTDKDGKPRVSNLRPGGLAARSDLLNIGDYIRSVNGIHLTRLRHDEIITLLKNVGERVVLE VEY 201 KIAA1719 domain 31267982 IQIVHTETTEVVLCGDPLSGFGLQLQGGIFATETLSSPPLVCFIEPDSPAERCGLLQVGDRVLSINGIATEDGTMEEANQL LRDAALAHKVVLEVEFDVAESV 202KIAA1719 domain 1 1267982 ILDVSLYKEGNSFGFVLRGGAHEDGHKSRPLVLTYVRPGGPADREGSLKVGDRLLSVDGIPLHGASHATALATLRQCS HEALFQVEYDVATP 203 KIAA1719domain 4 1267982 QFDVAESVIPSSGTFHVKLPKKRSVELGITISSASRKRGEPLIISDIKKGSVAHRTGTLEPGDKLLAIDNIRLDNCPMEDA VQILRQCEDLVKLKIRKDEDN 204 LIMmystique 12734250 MALTVDVAGPAPWGFRITGGRDFHTPIMVTKVAERGKA domain 1KDADLRPGDIIVAINGESAEGMLHAEAQSKIRQSPSPLRL QLDRSQATSPGQT 205 LIM proteindomain 1 3108092 SNYSVSLVGPAPWGFRLQGGKDFNMPLTISSLKDGGKAAQANVRIGDVVLSIDGINAQGMTHLEAQNKIKGCTGSLNM TLQRAS 206 LIMK1 domain 14587498 TLVEHSKLYCGHCYYQTVVTPVIEQILPDSPGSHLPHTVTLVSIPASSHGKRGLSVSIDPPHGPPGCGTEHSHTVRVQGVDPGCMSPDVKNSIHVGDRILEINGTPIRNVPLDEIDLLIQE TSRLLQLTLEHD 207 LIMK2 domain1 1805593 PYSVTLISMPATTEGRRGFSVSVESACSNYATTVQVKEVNRMHISPNNRNAIHPGDRILEINGTPVRTLRVEEVEDAISQ TSQTLQLLIEHD 208 LIM-RILdomain 1 1085021 IHSVTLRGPSPWGFRLVGRDFSAPLTISRVHAGSKASLAALCPGDLIQAINGESTELMTHLEAQNRIKGCHDHLTLSVSR PE 209 LU-1 domain 1 U52111VCYRTDDEEDLGIYVGEVNPNSIAAKDGRIREGDRIIQINGVDVQNREEAVAILSQEENTNISLLVARPESQLA 210 MAGI 1 domain 2 3370997IPATQPELITVHIVKGPMGFGFTIADSPGGGGQRVKQIVDSPRCRGLKEGDLIVEVNKKNVQALTHNQVVDMLVECPKG SEVTLLVQRGGNSS 211 MAGI 1 domain5 3370997 IPDYQEQDIFLWRKETGFGFRILGGNEPGEPIYIGHIVPLGAADTDGRLRSGDELICVDGTPVIGKSHQLVVQLMQQAA KQGHVNLTVRRKVVFAVPKTENSS 212 MAGI1 domain 4 3370997 IPGVVSTVVQPYDVEIRRGENEGFGFVIVSSVSRPEAGTTFAGNACVAMPHKIGRIIEGSPADRCGKLKVGDRILAVNGCSITNKSHSDIVNLIKEAGNTVTLRIIPGDESSNAEFIVTD 213 MAGI 1 domain 1 3370997IPSELKGKFIHTKLRKSSRGFGFTVVGGDEPDEFLQIKSLVLDGPAALDGKMETGDVIVSVNDTCVLGHTHAQVVKIFQ SIPIGASVDLELCRGYPLPFDPDGIHRD 214MAGI 1 domain 3 3370997 QATQEQDFYTVELERGAKGFGFSLRGGREYNMDLYVLRLAEDGPAERCGKMRIGDEILEINGETTKNMKHSRAIELIK NGGRRVRLFLKRG 215 Magi 2 domain1 2947231 REKPLFTRDASQLKGTFLSTTLKKSNMGFGFTIIGGDEPDEFLQVKSVIPDGPAAQDGKMETGDVIVYINEVCVLGHTH ADVVKLFQSVPIGQSVNLVLCRGYP 216Magi 2 domain 3 2947231 HYKELDVHLRRMESGFGFRILGGDEPGQPILIGAVIAMGSADRDGRLHPGDELVYVDGIPVAGKTHRYVIDLMHHAAR NGQVNLTVRRKVLCG 217 Magi 2 domain4 2947231 EGRGISSHSLQTSDAVIHRKENEGFGFVIISSLNRPESGSTITVPHKIGRIIDGSPADRCAKLKVGDRILAVNGQSIINMPH ADIVKLIKDAGLSVTLRIIPQEEL 218Magi 2 domain 2 2947231 LSGATQAELMTLTIVKGAQGFGFTIADSPTGQRVKQILDIQGCPGLCEGDLIVEINQQNVQNLSHTEVVDILKDCPIGSE TSLIIHRGGFF 219 Magi 2 domain 52947231 LSDYRQPQDFDYFTVDMEKGAKGFGFSIRGGREYKMDLYVLRLAEDGPAIRNGRMRVGDQIIEINGESTRDMTHARAI ELIKSGGRRVRLLLKRGTGQ 220 Magi 2domain 6 2947231 HESVIGRNPEGQLGFELKGGAENGQFPYLGEVKPGKVAYESGSKLVSEELLLEVNETPVAGLTIRDVLAVIKHCKDPLR LKCVKQGGIHR 221 MAGI 3 domain 210047344 ASSGSSQPELVTIPLIKGPKGFGFAIADSPTGQKVKMILDSQWCQGLQKGDIIKEIYHQNVQNLTHLQVVEVLKQFPVG ADVPLLILRGGPPSPTKTAKM 222 MAGI 3domain 5 10047344 QNLGCYPVELERGPRGFGFSLRGGKEYNMGLFILRLAEDGPAIKDGRIHVGDQIVEINGEPTQGITHTRAIELIQAGGNK VLLLLRPGTGLIPDHGLA 223 MAGI 3domain 3 10047344 LYEDKPPNTKDLDVFLRKQESGFGFRVLGGDGPDQSIYIGAIIPLGAAEKDGRLRAADELMCIDGIPVKGKSHKQVLDL MTTAARNGHVLLTVRRKIFYGEKQPEDDS224 MAGI 3 domain 1 10047344 PSQLKGVLVRASLKKSTMGFGFTIIGGDRPDEFLQVKNVLKDGPAAQDGKIAPGDVIVDINGNCVLGHTHADVVQMF QLVPVNQYVNLTLCRGYPLPDDSED 225MAGI 3 domain 4 10047344 PAPQEPYDVVLQRKENEGFGFVILTSKNKPPPGVIPHKIGRVIEGSPADRCGKLKVGDHISAVNGQSIVELSHDNIVQLI KDAGVTVTLTVIAEEEHHGPPS 226MAST1 domain 1 4589589 GLRSPITIQRSGKKYGFTLRAIRVYMGDTDVYSVHHIVWHVEEGGPAQEAGLCAGDLITHVNGEPVHGMVHPEVVELI LKSGNKVAVTTTPFEN 227 MAST2domain 1 3882334 ISALGSMRPPIIIHRAGKKYGFTLRAIRVYMGDSDVYTVHHMVWHVEDGGPASEAGLRQGDLITHVNGEPVHGLVHTE VVELILKSGNKVAISTTPLENSS 228 MAST3domain 1 3043645 LCGSLRPPIVIHSSGKKYGFSLRAIRVYMGDSDVYTVHHVVWSVEDGSPAQEAGLRAGDLITHINGESVLGLVHMDVV ELLLKSGNKISLRTTALENTSIKVG 229MAST4 domain 1 2224546 PHQPIVIHSSGKNYGFTIRAIRVYVGDSDIYTVHHIVWNVEEGSPACQAGLKAGDLITHINGEPVHGLVHTEVIELLLKS GNKVSITTTPF 230 MGC5395 domain1 BC012477 PAKMEKEETTRELLLPNWQGSGSHGLTIAQRDDGVFVQEVTQNSPAARTGVVKEGDQIVGATIYFDNLQSGEVTQLLN TMGHHTVGLKLHRKGDRSPNSS 231 MINT1domain 1 2625024 SENCKdVFIEKQKGEILGVVIVESGWGSILPTVIIANMMHGGPAEKSGKLNIGDQIMSINGTSLVGLPLSTCQSIIKGLKN QSRVKLNIVRCPPVNSS 232 MINT1domains 1 2625024 SENCKDVFIEKQKGEILGVVIVESGWGSILPTVIIANMMH and 2GGPAEKSGKLNIGDQIMSINGTSLVGLPLSTCQSIIKGLENQSRVKLNIVRCPPVTTVLIRRPDLRYQLGFSVQNGIICSLMRGGIAERGGVRVGHRIIEINGQSVVATPHEKIVHILSNAV GEIHMKTMPAAMYRLL 233 MINT1domain 2 2625024 LRCPPVTTVLIRRPDLRYQLGFSVQNGIICSLMRGGIAERGGVRVGHRIIEINGQSVVATPHEKIVHILSNAVGEIHMKTM PAAMYRLLNSS 234 MINT3 domain 13169808 HNGDLDHFSNSDNCREVHLEKRRGEGLGVALVESGWGSLLPTAVIANLLHGGPAERSGALSIGDRLTAINGTSLVGLPL AACQAAVRETKSQTSVTLSIVHCPPVT235 MINT3 domain 2 3169808 PVTTAIIHRPHAREQLGFCVEDGIICSLLRGGIAERGGIRVGHRIIEINGQSVVATPHARIIELLTEAYGEVHIKTMPAATY RLLTGNSS 236 MINT3 domain 13169808 LSNSDNCREVHLEKRRGEGLGVALVESGWGSLLPTAVIANLLHGGPAERSGALSIGDRLTAINGTSLVGLPLAACQAA VRETKSQTSVTLSIVHCPPVTTAIM 237MPP1 domain 1 189785 RKVRLIQFEKVTEEPMGITLKLNEKQSCTVARILHGGMIHRQGSLHVGDEILEINGTNVTNHSVDQLQKAMKETKGMIS LKVIPNQ 238 MPP2 domain 1 939884PVPPDAVRMVGIRKTAGEHLGVTFRVEGGELVIARILHGGMVAQQGLLHVGDIIKEVNGQPVGSDPRALQELLRNAS GSVILKILPNYQ 239 MPP3 domain 121536463 NIDEDFDEESVKIVRLVKNKEPLGATIRRDEHSGAVVVARIMRGGAADRSGLVHVGDELREVNGIAVLHKRPDEISQI LAQSQGSITLKIIPATQEEDR 240 MUPP1domain 5 2104784 WEAGIQHIELEKGSKGLGFSILDYQDPIDPASTVIIIRSLVPGGIAEKDGRLLPGDRLMFVNDVNLENSSLEEAVEALKG APSGTVRIGVAKPLPLSPEE 241 MUPP1domain 12 2104784 LQGLRTVEMKKGPTDSLGISIAGGVGSPLGDVPIFIAMMHPTGVAAQTQKLRVGDRIVTICGTSTEGMTHTQAVNLLKN ASGSIEMQVVAGGDVSV 242 MUPP1domain 2 2104784 PVHWQHMETIELVNDGSGLGFGIIGGKATGVIVKTILPGGVADQHGRLCSGDHILKIGDTDLAGMSSEQVAQVLRQCG NRVKLMIARGAIEERTAPT 243 MUPP1domain 3 2104784 QESETFDVELTKNVQGLGITIAGYIGDKKLEPSGIFVKSITKSSAVEHDGRIQIGDQIIAVDGTNLQGFTNQQAVEVLRH TGQTVLLTLMRRGMKQEA 244 MUPP1domain 11 2104784 KEEEVCDTLTIELQKKPGKGLGLSIVGKRNDTGVFVSDIVKGGIADADGRLMQGDQILMVNGEDVRNATQEAVAALL KCSLGTVTLEVGRIKAGPFHS 245 MUPP1domain 8 2104784 LTGELHMIELEKGHSGLGLSLAGNKDRSRMSVFIVGIDPNGAAGKDGRLQIADELLEINGQILYGRSHQNASSIIKCAPS KVKIIFIRNKDAVNQ 246 MUPP1domain 13 2104784 LGPPQCKSITLERGPDGLGFSIVGGYGSPHGDLPIYVKTVFAKGAASEDGRLKRGDQIIAVNGQSLEGVTHEEAVAILKR TKGTVTLMVLS 247 MUPP1 domain 62104784 RNVSKESFERTINIAKGNSSLGMTVSANKDGLGMIVRSIIHGGAISRDGRIAIGDCILSINEESTISVTNAQARAMLRRHS LIGPDIKITYVPAEHLEE 248 MUPP1domain 10 2104784 LPGCETTIEISKGRTGLGLSIVGGSDTLLGAIIIHEVYEEGAACKDGRLWAGDQILEVNGIDLRKATHDEAINVLRQTPQR VRLTLYRDEAPYKE 249 MUPP1 domain7 2104784 LNWNQPRRVELWREPSKSLGISIVGGRGMGSRLSNGEVMRGIFIKHVLEDSPAGKNGTLKPGDRIVEVDGMDLRDASHEQAVEAIRKAGNPVVFMVQSIINRPRKSPLPSLL 250 MUPP1 domain 9 2104784LSSFKNVQHLELPKDQGGLGIAISEEDTLSGVIIKSLTEHGVAATDGRLKVGDQILAVDDEIVVGYPIEKFISLLKTAKM TVKLTIHAENPDSQ 251 MUPP1 domain1 2104784 QGRHVEVFELLKPPSGGLGFSVVGLRSENRGELGIFVQEIQEGSVAHRDGRLKETDQILAINGQALDQTITHQQAISILQ KAKDTVQLVIARGSLPQLV 252 MUPP1domain 4 2104784 LNYEIVVAHVSKFSENSGLGISLEATVGHHFIRSVLPEGPVGHSGKLFSGDELLEVNGITLLGENHQDVVNILKELPIEVT MVCCRRTVPPT 253 NeDLG domain 210863920 ITLLKGPKGLGFSIAGGIGNQHIPGDNSIYITKIIEGGAAQKDGRLQIGDRLLAVNNTNLQDVRHEEAVASLKNTSDMVY LKVAKPGSLE 254 NeDLG domain 110863920 IQYEEIVLERGNSGLGFSIAGGIDNPHVPDDPGIFITKIIPGGAAAMDGRLGVNDCVLRVNEVEVSEVVHSRAVEALKEA GPVVRLVVRRRQN 255 NeDLG domain 310863920 ILLHKGSTGLGFNIVGGEDGEGIFVSFILAGGPADLSGELRRGDRILSVNGVNLRNATHEQAAAALKRAGQSVTIVAQYRPEEYSRFESKIHDLREQMMNSSMSSGSGSLRTSEKRSLE 256 NeDLG domains 1 10863920YEEIVLERGNSGLGFSIAGGIDNPHVPDDPGIFITKIIPGGA and 2AAMDGRLGVNDCVLRVNEVEVSEVVHSRAVEALKEAGPVVRLVVRRRQPPPETIMEVNLLKGPKGLGFSIAGGIGNQHIPGDNSIYITKIIEGGAAQKDGRLQIGDRLLAVNNTNLQ DVRHEEAVASLKNTSDMVYLKVAKPGSL257 Neurabin II domain 1 AJ401189RVERLELFPVELEKDSEGLGISIIGMGAGADMGLEKLGIFVKTVTEGGAAHRDGRIQVNDLLVEVDGTSLVGVTQSFA ASVLRNTKGRVRCRFMIGRERPGEQSEV 258NOS1 domain 1 642525 QPNVISVRLFKRKVGGLGFLVKERVSKPPVIISDLIRGGAAEQSGLIQAGDIILAVNGRPLVDLSYDSALEVLRGIASET HVVLILRGPE 259 novel PDZ gene7228177 PSDTSSEDGVRRIVHLYTTSDDFCLGFNIRGGKEFGLGIY domain 2VSKVDHGGLAEENGIKVGDQVLAANGVRFDDISHSQAV EVLKGQTHIMLTIKETGRYPAYKEM 260novel PDZ gene 7228177 EANSDESDIIHSVRVEKSPAGRLGFSVRGGSEHGLGIFVS domain 1KVEEGSSAERAGLCVGDKITEVNGLSLESTTMGSAVKVL TSSSRLHMMVRRMGRVPGIKFSKEK 261novel serine 1621243 DKIKKFLTESHDRQAKGKAITKKKYIGIRMMSLTSSKAK proteasedomain 1 ELKDRHRDFPDVISGAYIIEVIPDTPAEAGGLKENDVIISINGQSVVSANDVSDVIKRESTLNMVVRRGNEDIMITV 262 Numb BP domain 2 AK056823YRPRDDSFHVILNKSSPEEQLGIKLVRKVDEPGVFIFNALDGGVAYRHGQLEENDRVLAINGHDLRYGSPESAAHLIQ ASERRVHLVVSRQVRQRSPD 263 Numb BPdomain 3 AK056823 PTITCHEKVVNIQKDPGESLGMTVAGGASHREWDLPIYVISVEPGGVISRDGRIKTGDILLNVDGVELTEVSRSEAVALL KRTSSSIVLKALEVKEYEPQ 264 NumbBP domain 1 AK056823 PDGEITSIKINRVDPSESLSIRLVGGSETPLVHIIIQHIYRDGVIARDGRLLPRDIILKVNGMDISNVPHNYAVRLLRQPCQ VLWLTVMREQKFRSR 265 Numb BPdomain 4 AK056823 PRCLYNCKDIVLRRNTAGSLGFCIVGGYEEYNGNKPFFIKSIVEGTPAYNDGRIRCGDILLAVNGRSTSGMIHACLARLL KELKGRITLTIVSWPGTFL 266 outermembrane 7023825 LLTEEEINLTRGPSGLGFNIVGGTDQQYVSNDSGIYVSRIK domain 1ENGAAALDGRLQEGDKILSVNGQDLKNLLHQDAVDLFR NAGYAVSLRVQHRLQVQNGIHS 267 p55Tdomain 1 12733367 PVDAIRILGIHKRAGEPLGVTFRVENNDLVIARILHGGMIDRQGLLHVGDIIKEVNGHEVGNNPKELQELLKNISGSVT LKILPSYRDTITPQQ 268 PAR3 domain2 8037914 GKRLNIQLKKGTEGLGFSITSRDVTIGGSAPIYVKNILPRGAAIQDGRLKAGDRLIEVNGVDLVGKSQEEVVSLLRSTKM EGTVSLLVFRQEDA 269 PAR3 domain 18037914 IPNFSLDDMVKLVEVPNDGGPLGIHVVPFSARGGRTLGLLVKRLEKGGKAEHENLFRENDCIVRINDGDLRNRRFEQA QHMFRQAMRTPIIWFHVVPAANKEQYEQ 270PAR3 domain 3 8037914 PREFLTFEVPLNDSGSAGLGVSVKGNRSKENHADLGIFVKSIINGGAASKDGRLRVNDQLIAVNGESLLGKTNQDAMETLRRSMSTEGNKRGMIQLIVASRISKCNELKSNSS 271 PAR3L domain 2 18874467ISNKNAKKIKIDLKKGPEGLGFTVVTRDSSIHGPGPIFVKNILPKGAAIKDGRLQSGDRILEVNGRDVTGRTQEELVAML RSTKQGETASLVIARQEGH 272 PAR3Ldomain 3 18874467 ITSEQLTFEIPLNDSGSAGLGVSLKGNKSRETGTDLGIFIKSIIHGGAAFKDGRLRMNDQLIAVNGESLLGKSNHEAMETL RRSMSMEGNIRGMIQLVILRRPERP 273PAR3L domain 1 18874467 IPRTKDTLSDMTRTVEISGEGGPLGIHVVPFFSSLSGRILGLFIRGIEDNSRSKREGLFHENECIVKINNVDLVDKTFAQAQ DVFRQAMKSPSVLLHVLPPQNR 274 PAR6domain 1 2613011 PETHRRVRLHKHGSDRPLGFYIRDGMSVRVAPQGLERVPGIFISRLVRGGLAESTGLLAVSDEILEVNGIEVAGKTLDQ VTDMMVANSHNLIVTVKPANQRNNV 275PAR6 beta domain 1 1353716 IPVSSIIDVDILPETHRRVRLYKYGTEKPLGFYIRDGSSVRVTPHGLEKVPGIFISRLVPGGLAQSTGLLAVNDEVLEVNGIEVSGKSLDQVTDMMIANSRNLIITVRPANQRNNRIHRD 276 PAR6 GAMMA 13537118IDVDLVPETHRRVRLHRHGCEKPLGFYIRDGASVRVTPH domain 1GLEKVPGIFISRMVPGGLAESTGLLAVNDEVLEVNGIEVAGKTLDQVTDMMIANSHNLIVTVKPANQRNNVV 277 PDZ-73 domain 3 5031978PEQIMGKDVRLLRIKKEGSLDLALEGGVDSPIGKVVVSAVYERGAAERHGGIVKGDEIMAINGKIVTDYTLAEADAAL QKAWNQGGDWIDLVVAVCPPKEYDD 278PDZ-73 domain 2 5031978 IPGNRENKEKKVFISLVGSRGLGCSISSGPIQKPGIFISHVKPGSLSAEVGLEIGDQIVEVNGVDFSNLDHKEAVNVLKSS RSLTISIVAAAGRELFMTDEF 279 PDZ-73domain 1 5031978 RSRKLKEVRLDRLHPEGLGLSVRGGLEFGCGLFISHLIKGGQADSVGLQVGDEIVRINGYSISSCTHEEVINLIRTKKTVS IKVRHIGLIPVKSSPDEFH 280 PDZK1domain 2 2944188 RLCYLVKEGGSYGFSLKTVQGKKGVYMTDITPQGVAMRAGVLADDHLIEVNGENVEDASHEEVVEKVKKSGSRVMF LLVDKETDKREFIVTD 281 PDZK1 domain3 2944188 QFKRETASLKLLPHQPRIVEMKKGSNGYGFYLRAGSEQKGQIIKDIDSGSPAEEAGLKNNDLVVAVNGESVETLDHDSVVEMIRKGGDQTSLLVVDKETDNMYRLAEFIVTD 282 PDZK1 domains 2 2944188RLCYLVKEGGSYGFSLKTVQGKKGVYMTDITPQGVAMR and 3 and 4AGVLADDHLIEVNGENVEDASHEKVVEKVKKSGSRVMFLLVDKETDKRHVEQKIQFKRETASLKLLPHQPRIVEMKKGSNGYGFYLRAGSEQKGQIIKDIDSGSPAEEAGLKNNDLVVAVNGESVETLDHDSVVEMIRKGGDQTSLLVVDKETDNMYRLAHFSPFLYYQSQELPNGSVKEAPAPTPTSLEVSSPPDTTEEVDHKPKLCRLAKGENGYGFHLNAIRGLPGSFIKEVQKGGPADLAGLEDEDVIIEVNGVNVLDEPYEKVVDRIQ SSGKNVTLLVCGK 283 PDZK1 domain 42944188 PDTTEEVDHKPKLCRLAKGENGYGFHLNAIRGLPGSFIKEVQKGGPADLAGLEDEDVIIEVNGVNVLDEPYEKVVDRIQ SSGKNVTLLVGKNSS 284 PDZK1 domain1 2944188 LTSTFNPRECKLSKQEGQNYGFFLRIEKDTEGHLVRVVEKCSPAEKAGLQDGDRVLRINGVFVDKEEHMQVVDLVRK SGNSVTLLVLDGDSYEKAGSHEPS 285PICK1 domain 1 4678411 LGIPTVPGKVTLQKDAQNLIGISIGGGAQYCPCLYIVQVFDNTPAALDGTVAAGDEITGVNGRSIKGKTKVEVAKMIQE VKGEVTIHYNKLQADPKQGM 286 PISTdomain 1 98374330 SQGVGPIRKVLLLKEDHEGLGISITGGKEHGVPILISEIHPGQPADRCGGLHVGDAILAVNGVNLRDTKHKEAVTILSQQ RGEIEFEVVYVAPEVDSD 287 prIL16domain 2 1478492 TAEATVCTVTLEKMSAGLGFSLEGGKGSLHGDKPLTINRIFKGAASEQSETVQPGDEILQLGGTAMQGLTRFEAWNIIK ALPDGPVTIVIRRKSLQSK 288 prIL16domain 1 1478492 IHVTILHKEEGAGLGFSLAGGADLENKVITVHRVFPNGLASQEGTIQKGNEVLSINGKSLKGTTHHDALAILRQAREPRQ AVIVTRKLTPEEFIVTD 289 prIL16domains 1 1478492 IHVTILHKEEGAGLGFSLAGGADLENKVITVHRVFPNGLA and 2SQEGTIQKGNEVLSINGKSLKGTTHHDALAILRQAREPRQAVIVTRKLTPEAMPDLNSSTDSAASASAASDVSVESTAEATVCTVTLEKMSAGLGFSLEGGKGSLHGDKPLTINRIFKGAASEQSETVQPGDEILQLGGTAMQGLTRFEAWNIIKAL PDGPVTIVIRRKSLQSK 290 PSAPdomain 1 6409315 IREAKYSGVLSSIGKIFKEEGLLGFFVGLIPHLLGDVVFLWGCNLLAHFINAYLVDDSVSDTPGGLGNDQNPGSQFSQALAIRSYTKFVMGIAVSMLTYPFLLVGDLMAVNNCGLQAGLPPYSPVFKSWIHCWKYLSVQGQLFRGSSLLFRRVSSGSC FALE 291 PSD95 domains 13318652 EGEMEYEEITLERGNSGLGFSIAGGTDNPHIGDDPSIFITKI and 2 and 3IPGGAAAQDGRLRVNDSILFVNEVDVREVTHSAAVEALKEAGSIVRLYVMRRKPPAEKVMEIKLIKGPKGLGFSIAGGVGNQHIPGDNSIYVTKIIEGGAAHKDGRLQIGDKILAVNSVGLEDVMHEDAVAALKNTYDVVYLKVAKPSNAYLSDSYAPPDITTSYSQHLDNEISHSSYLGTDYPTAMTPTSPRRYSPVAKDLLGEEDIPREPRRIVIHRGSTGLGFNIVGGEDGEGIFISFILAGGPADLSGELRKGDQILSVNGVDLRNASHEQAAI ALKNAGQTVTIIAQYKPE 292 PSD95domain 2 3318652 HVMRRKPPAEKVMEIKLIKGPKGLGFSIAGGVGNQHIPGDNSIYVTKIIEGGAAHKDGRLQIGDKILAVNSVGLEDVM HEDAVAALKNTYDVVYLKVAKPSNAYL 293PSD95 domain 3 3318652 REDIPREPRRIVIHRGSTGLGFNIVGGEDGEGIFISFILAGGPADLSGELRKGDQILSVNGVDLRNASHEQAAIALKNAGQ TVTIIAQYKPEFIVTD 294 PSD95domain 1 3318652 LEYEeITLERGNSGLGFSIAGGTDNPHIGDDPSIFITKIIPGGAAAQDGRLRVNDSILFVNEVDVREVTHSAAVEALKEAG SIVRLYVMRRKPPAENSS 295 PSMD9domain 1 9184389 RDMAEAHKEAMSRKLGQSESQGPPRAFAKVNSISPGSPASIAGLQVDDEIVEFGSVNTQNFQSLHNIGSVVQHSEGALA PTILLSVSM 296 PTN-3 domain 1179912 QNDNGDSYLVLIRITPDEDGKFGFNLKGGVDQKMPLVVSRINPESPADTCIPKLNEGDQIVLINGRDISEHTHDQVVMFI KASRESHSRELALVIRRRAVRS 297PTN-4 domain 1 190747 IRMKPDENGRFGFNVKGGYDQKMPVIVSRVAPGTPADLCVPRLNEGDQVVLINGRDIAEHTHDQVVLFIKASCERHS GELMLLVRPNA 298 PTPL1 domain 2515030 GDIFEVELAKNDNSLGISVTGGVNTSVRHGGIYVKAVIPQGAAESDGRIHKGDRVLAVNGVSLEGATHKQAVETLRNT GQVVHLLLEKGQSPTSK 299 PTPL1domain 1 515030 PEREITLVNLKKDAKYGLGFQIIGGEKMGRLDLGIFISSVAPGGPADFHGCLKPGDRLISVNSVSLEGVSHHAAIEILQNA PEDVTLVISQPKEKISKVPSTPVHL 300PTPL1 domain 4 515030 ELEVELLITLIKSEKASLGFTVTKGNQRIGCYVHDVIQDPAKSDGRLKPGDRLIKVNDTDVTNMTHTDAVNLLRAASK TVRLVIGRVLELPRIPMLPH 301 PTPL1domain 3 515030 TEENTFEVKLFKNSSGLGFSFSREDNLIPEQINASIVRVKKLFAGQPAAESGKIDVGDVILKVNGASLKGLSQQEVISALR GTAPEVFLLLCRPPPGVLPEIDT 302PTPL1 domain 5 515030 MLPHLLPDITLTCNKEELGFSLCGGHDSLYQVVYISDINPRSVAAIEGNLQLLDVIHYVNGVSTQGMTLEEVNRALDM SLPSLVLKATRNDLPV 303 RGS 3 domain1 18644735 VCSERRYRQITIPRGKDGFGFTICCDSPVRVQAVDSGGPAERAGLQQLDTVLQLNERPVEHWKCVELAHEIRSCPSEIIL LVWRMVPQVKPG 304 RGS12 domain 13290015 RPSPPRVRSVEVARGRAGYGFTLSGQAPCVLSCVMRGSPADFVGLRAGDQILAVNEINVKKASHEDVVKLIGKCSGVL HMVIAEGVGRFESCS 305 Rho-GAP 1050345878 SEDETFSWPGPKTVTLKRTSQGFGFTLRHFIVYPPESAIQF domain 1SYKDEENGNRGGKQRNRLEPMDTIFVKQVKEGGPAFEAGLCTGDRIIKVNGESVIGKTYSQVIALIQNSDTTLELSVMP KDED 306 Rhophilin domain 1AY082588 SAKNRWRLVGPVHLTRGEGGFGLTLRGDSPVLIAAVIPGSQAAAAGLKEGDYIVSVNGQPCRWWRHAEVVTELKAA GEAGASLQVVSLLPSSRLPS 307Rhophilin-like AF268032 ISFSANKRWTPPRSIRFTAEEGDLGFTLRGNAPVQVHFLD domain1 PYCSASVAGAREGDYIVSIQLVDCKWLTLSEVMKLLKSF GEDEIEMKVVSLLDSTSSMHNKSAT 308RIM2 domain 1 12734165 TLNEEHSHSDKHPVTWQPSKDGDRLIGRILLNKRLKDGSVPRDSGAMLGLKVVGGKMTESGRLCAFITKVKKGSLADTVGHLRPGDEVLEWNGRLLQGATFEEVYNIILESKPEPQ VELVVSRPIG 309 SEMCAP 3 domain 25889526 QEMDREELELEEVDLYRMNSQDKLGLTVCYRTDDEDDIGIYISEIDPNSIAAKDGRIREGDRIIQINGIEVQNREEAVAL LTSEENKNFSLLIARPELQLD 310SEMCAP 3 domain 1 5889526 QGEETKSLTLVLHRDSGSLGFNIIGGRPSVDNHDGSSSEGIFVSKIVDSGPAAKEGGLQIHDRIIEVNGRDLSRATHDQAV EAFKTAKEPIVVQVLRRTPRTKMFTP 311semcap2 domain 1 7019938 ILAHVKGIEKEVNVYKSEDSLGLTITDNGVGYAFIKRIKDGGVIDSVKTICVGDHIESINGENIVGWRHYDVAKKLKEL KKEELFTMKLIEPKKAFEI 312 serineprotease 2738914 RGEKKNSSSGISGSQRRYIGVMMLTLSPSILAELQLREPSF domain 1PDVQHGVLIHKVILGSPAHRAGLRPGDVILAIGEQMVQN AEDVYEAVRTQSQLAVQIRRGRETLTLYV313 Shank 1 domain 1 6049185 ILEEKTVVLQKKDNEGFGFVLRGAKADTPIEEFTPTPAFPALQYLESVDEGGVAWQAGLRTGDFLIEVNNENVVKVGH RQVVNMIRQGGNHLVLKVVTVTRNLDPDDNSS314 Shank 2 domain 1 7025450 ILKEKTVLLQKKDSEGFGFVLRGAKAQTPIEEFTPTPAFPALQYLESVDEGGVAWRAGLRMGDFLIEVNGQNVVKVGHRQVVNMIRQGGNTLMVKVVMVTRHPDMDEAVQNSS 315 Shank 3 domain 1 *SDYVIDDKVAVLQKRDHEGFGFVLRGAKAETPIEEFTPTPAFPALQYLESVDVEGVAWRAGLRTGDFLIEVNGVNVVK VGHKQVVALIRQGGNRLVMKVVSVTRKPEEDG316 sim to lig of numb 22477649SNSPREEIFQVALHKRDSGEQLGIKLVRRTDEPGVFILDLL px2 domain 2EGGLAAQDGRLSSNDRVLAINGHDLKYGTPELAAQIIQA SGERVNLTIARPGKPQPG 317 sim tolig of numb 22477649 IQCVTCQEKHITVKKEPHESLGMTVAGGRGSKSGELPIFV px2 domain3 TSVPPHGCLARDGRIKRGDVLLNINGIDLTNLSHSEAVA MLKASAASPAVALKALEVQIVEEAT 318Similar to 14286261 MGLGVSAEQPAGGAEGFHLHGVQENSPAQQAGLEPYFD GRASP65domain 1 FIITIGHSRLNKENDTLKALLKANVEKPVKLEVFNMKTMRVREVEVVPSNMWGGQGLLGASVRFCSFRRASE 319 Similar to 14286261RASEQVWHVLDVEPSSPAALAGLRPYTDYVVGSDQILQE GRASP65 domain 2SEDFFTLIESHEGKPLKLMVYNSKSDSCRESGMWHWLW VSTPDPNSAPQLPQEATWHPTTFCSTTWCPTT320 Similar to Protein- 21595065 ISVTDGPKFEVKLKKNANGLGFSFVQMEKESCSHLKSDLTyrosine- VRIKRLFPGQPAEENGAIAAGDIILAVNGRSTEGLIFQEVL PhosphataseHLLRGAPQEVTLLLCRPPPGA Homolog domain 1 321 SIP1 domain 1 2047327QPEPLRPRLCRLVRGEQGYGFHLHGEKGRRGQFIRRVEPGSPAEAAALRAGDRLVEVNGVNVEGETHHQVVQRIKAV EGQTRLLVVDQETDEELRRRNSS 322 SIP1domain 2 2047327 PLRELRPRLCHLRKGPQGYGFNLHSDKSRPGQYIRSVDPGSPAARSGLRAQDRLIEVNGQNVEGLRHAEVVASIKARE DEARLLVVDPETDEHFKRNSS 323 SITAC18 domain 1 8886071 PGVREIHLCKDERGKTGLRLRKVDQGLFVQLVQANTPASLVGLRFGDQLLQIDGRDCAGWSSHKAHQVVKKASGDKI VVVVRDRPFQRTVTM 324 SITAC 18domain 2 8886071 PFQRTVTMHKDSMGHVGFVIKKGKIVSLVKGSSAARNGLLTNHYVCEVDGQNVIGLKDKKIMEILATAGNVVTLTIIP SVIYEHIVEFIV 325 SNPC IIadomain 1 20809633 SLERPRFCLLSKEEGKSFGFHLQQELGRAGHVVCRVDPGTSAQRQGLQEGDRILAVNNDVVEHEDYAVVVRRIRASSP RVLLTVLARHAHDVARAQ 326 SYNTENINdomain 2 2795862 LRDRPFERTITMHKDSTGHVGFIFKNGKITSIVKDSSAARNGLLTEHNICEINGQNVIGLKDSQIADILSTSGTVVTITIMP AFIFEHMNSS 327 SYNTENINdomain 1 2795862 LEIKQGIREVILCKDQDGKIGLRLKSIDNGIFVQLVQANSPASLVGLRFGDQVLQINGENCAGWSSDKAHKVLKQAFGE KITMRIHRD 328 Syntrophin 1 alpha1145727 QRRRVTVRKADAGGLGISIKGGRENKMPILISKIFKGLAA domain 1DQTEALFVGDAILSVNGEDLSSATHDEAVQVLKKTGKEV VLEVKYMKDVSPYFK 329 Syntrophinbeta 2 476700 PVRRVRVVKQEAGGLGISIKGGRENRMPILISKIFPGLAA domain 1DQSRALRLGDAILSVNGTDLRQATHDQAVQALKRAGKE VLLEVKFIRE 330 Syntrophin gamma9507162 EPFYSGERTVTIRRQTVGGFGLSIKGGAEHNIPVVVSKISK 1 domain 1EQRAELSGLLFIGDAILQINGINVRKCRHEEVVQVLRNAG EEVTLTVSFLKRAPAFLKL 331Syntrophin gamma 9507164 SHQGRNRRTVTLRRQPVGGLGLSIKGGSEHNVPVVISKIF 2domain 1 EDQAADQTGMLFVGDAVLQVNGIHVENATHEEVVHLLR NAGDEVTITVEYLREAPAFLK332 TAX2-like protein 3253116 RGETKEVEVTKTEDALGLTITDNGAGYAFIKRIKEGSIINRdomain 1 IEAVCVGDSIEAINDHSIVGCRHYEVAKMLRELPKSQPFT LRLVQPKRAF 333 TIAM1domain 1 4507500 HSIHIEKSDTAADTYGFSLSSVEEDGIRRLYVNSVKETGLASKKGLKAGDEILEINNRAADALNSSMLKDFLSQPSLGL LVRTYPELE 334 TIAM2 domain 16912703 PLNVYDVQLTKTGSVCDFGFAVTAQVDERQHLSRIFISDVLPDGLAYGEGLRKGNEIMTLNGEAVSDLDLKQMEALF SEKSVGLTLIARPPDTKATL 335 TIP1domain 1 2613001 QRVEIHKLRQGENLILGFSIGGGIDQDPSQNPFSEDKTDKGIYVTRVSEGGPAEIAGLQIGDKIMQVNGWDMTMVTHD QARKRLTKRSEEVVRLLVTRQSLQK 336TIP2 domain 1 2613003 RKEVEVFKSEDALGLTITDNGAGYAFIKRIKEGSVIDHIHLISVGDMIEAINGQSLLGCRHYEVARLLKELPRGRTFTLKL TEPRK 337 TIP33 domain 12613007 HSHPRVVELPKTDEGLGFNVMGGKEQNSPIYISRIIPGGVAERHGGLKRGDQLLSVNGVSVEGEHHEKAVELLKAAKD SVKLVVRYTPKVL 338 TIP43 domain 12613011 LSNQKRGVKVLKQELGGLGISIKGGKENKMPILISKIFKGLAADQTQALYVGDAILSVNGADLRDATHDEAVQALKRA GKEVLLEVKYMREATPYVK 339 UnknownPDZ 22382223 IQRSSIKTVELIKGNLQSVGLTLRLVQSTDGYAGHVIIETV domain 1APNSPAAIADLQRGDRLIAIGGVKITSTLQVLKLIKQAGD RVLVYYERPVGQSNQGA 340 Vartuldomain 1 1469875 ILTLTILRQTGGLGISIAGGKGSTPYKGDDEGIFISRVSEEGPAARAGVRVGDKLLEVNGVALQGAEHHEAVEALRGAG TAVQMRVWRERMVEPENAEFIVTD 341Vartul domain 4 1469875 RELCIQKAPGERLGISIRGGARGHAGNPRDPTDEGIFISKVSPTGAAGRDGRLRVGLRLLEVNQQSLLGLTHGEAVQLL RSVGDTLTVLVCDGFEASTDAALEVS 342Vartul domain 3 1469875 LEGPYPVEEIRLPRAGGPLGLSIVGGSDHSSHPFGVQEPGVFISKVLPRGLAARSGLRVGDRILAVNGQDVRDATHQEA VSALLRPCLELSLLVRRDPAEFIVTD 343Vartul domain 2 1469875 PLRQRHVACLARSERGLGFSIAGGKGSTPYRAGDAGIFVSRIAEGGAAHRAGTLQVGDRVLSINGVDVTEARHDHAVS LLTAASPTIALLLEREAGG 344 Vartuldomains 1 1469875 TLTILRQTGGLGISIAGGKGSTPYKGDDEGIFISRVSEEGP and 2AARAGVRVGDKLLEGIFVSRIAEGGAAHRAGTLQVGDRVLSINGVDVTEARHDHAVSLLTAASPTIALLLERE 345 X-11 beta domain 2 3005559IPPVTTVLIKRPDLKYQLGFSVQNGIICSLMRGGIAERGGVRVGHRIIEINGQSVVATAHEKIVQALSNSVGEIHMKTMPA AMFRLLTGQENSSL 346 X-11 betadomain 1 3005559 IHFSNSENCKELQLEKHKGEILGVVVVESGWGSILPTVILANMMNGGPAARSGKLSIGDQIMSINGTSLVGLPLATCQG IIKGLKNQTQVKLNIVSCPPVTTVLIKRNSS347 ZO-1 domain 1 292937 IWEQHTVTLHRAPGFGFGIAISGGRDNPHFQSGETSIVISDVLKGGPAEGQLQENDRVAMVNGVSMDNVEHAFAVQQL RKSGKNAKITIRRKKKVQIPNSS 348 ZO-1domain 2 292937 ISSQPAKPTKVTLVKSRKNEEYGLRLASHIFVKEISQDSLAARDGNIQEGDVVLKINGTVTENMSLTDAKTLIERSKGKL KMVVQRDRATLLNSS 349 ZO-1 domain3 292937 IRMKLVKFRKGDSVGLRLAGGNDVGIFVAGVLEDSPAAKEGLEEGDQILRVNNVDFTNIIREEAVLFLLDLPKGEEVTI LAQKKKDVFSN 350 ZO-2 domain 112734763 IQHTVTLHRAPGFGFGIAISGGRDNPHFQSGETSIVISDVLKGGPAEGQLQENDRVAMVNGVSMDNVEHAFAVQQLRK SGKNAKITIRRKKKVQIPNSS 351 ZO-2domain 3 12734763 HAPNTKMVRFKKGDSVGLRLAGGNDVGIFVAGIQEGTSAEQEGLQEGDQILKVNTQDFRGLVREDAVLYLLEIPKGE MVTILAQSRADVY 352 ZO-2 domain 212734763 RVLLMKSRANEEYGLRLGSQIFVKEMTRTGLATKDGNLHEGDIILKINGTVTENMSLTDARKLIEKSRGKLQLVVLRDS 353 ZO-3 domain 3 10092690RGYSPDTRVVRFLKGKSIGLRLAGGNDVGIFVSGVQAGSPADGQGIQEGDQILQVNDVPFQNLTREEAVQFLLGLPPG EEMELVTQRKQDIFWKMVQSEFIVTD 354ZO-3 domain 1 10092690 IPGNSTIWEQHTATLSKDPRRGFGIAISGGRDRPGGSMVVSDVVPGGPAEGRLQTGDHIVMVNGVSMENATSAFAIQIL KTCTKMANITVKRPRRIHLPAEFIVTD 355ZO-3 domain 2 10092690 QDVQMKPVKSVLVKRRDSEEFGVKLGSQIFIKHITDSGLAARHRGLQEGDLILQINGVSSQNLSLNDTRRLIEKSEGKL SLLVLRDRGQFLVNIPNSS *: No GInumber for this PDZ domain containing protein as it was computer clonedusing rat Shank3 sequence against human genomic clone AC000036 and insilico spliced together nucleotides 6400-6496, 6985-7109, 7211-7400 tocreate hypothetical human Shank3.

Methods for Detecting the Presence of an Oncogenic HPV E6 Protein in aSample

The invention provides a method of detecting the presence of anoncogenic HPV E6 protein in a sample. In general, the method involvescontacting a biological sample containing or potentially containing anoncogenic HPV E6 protein with a PDZ domain polypeptide and detecting anybinding of the oncogenic HPV E6 protein in said sample to the PDZ domainpolypeptide using a subject antibody. In alternative embodiments, asample may be contacted with a subject antibody, and the presence of theE6 protein may be detected using the PDZ domain polypeptide. In mostembodiments, binding of an oncogenic HPV E6 protein to the PDZ domainpolypeptide and a subject antibody indicates the presence of anoncogenic HPV E6 protein in the sample.

Biological samples to be analyzed using the methods of the invention maybe obtained from any mammal, e.g., a human or a non-human animal modelof HPV. In many embodiments, the biological sample is obtained from aliving subject.

In some embodiments, the subject from whom the sample is obtained isapparently healthy, where the analysis is performed as a part of routinescreening. In other embodiments, the subject is one who is susceptibleto HPV, (e.g., as determined by family history; exposure to certainenvironmental factors; etc.). In other embodiments, the subject hassymptoms of HPV (e.g., cervical warts, or the like). In otherembodiments, the subject has been provisionally diagnosed as having HPV(e.g. as determined by other tests based on e.g., PCR).

The biological sample may be derived from any tissue, organ or group ofcells of the subject. In some embodiments a cervical scrape, biopsy, orlavage is obtained from a subject. In other embodiments, the sample is ablood or urine sample.

In some embodiments, the biological sample is processed, e.g., to removecertain components that may interfere with an assay method of theinvention, using methods that are standard in the art. In someembodiments, the biological sample is processed to enrich for proteins,e.g., by salt precipitation, and the like. In certain embodiments, thesample is processed in the presence proteasome inhibitor to inhibitdegradation of the E6 protein.

In the assay methods of the invention, in some embodiments, the level ofE6 protein in a sample may be quantified and/or compared to controls.Suitable control samples are from individuals known to be healthy, e.g.,individuals known not to have HPV. Control samples can be fromindividuals genetically related to the subject being tested, but canalso be from genetically unrelated individuals. A suitable controlsample also includes a sample from an individual taken at a time pointearlier than the time point at which the test sample is taken, e.g., abiological sample taken from the individual prior to exhibiting possiblesymptoms of HPV.

In certain embodiments, a sample is contacted to a solid support-boundPDZ domain polypeptide under conditions suitable for binding of the PDZdomain polypeptide to any PL proteins in the sample, and, afterseparation of unbound sample proteins from the bound proteins, the boundproteins are detected using the subject antibody using known methods.

Kits

The present invention also includes kits for carrying out the methods ofthe invention. A subject kit usually contains a subject antibody. Inmany embodiments, the kits contain a first and second binding partner,where the first binding partner is a PDZ domain polypeptide and thesecond binding partner is a subject antibody. In some embodiments, thesecond binding partner is labeled with a detectable label. In otherembodiments, a secondary labeling component, such as a detectablylabeled secondary antibody, is included. In some embodiments, a subjectkit further comprises a means, such as a device or a system, forisolating oncogenic HPV E6 from the sample. The kit may optionallycontain proteasome inhibitor.

A subject kit can further include, if desired, one or more of variousconventional components, such as, for example, containers with one ormore buffers, detection reagents or antibodies. Printed instructions,either as inserts or as labels, indicating quantities of the componentsto be used and guidelines for their use, can also be included in thekit. In the present disclosure it should be understood that thespecified materials and conditions are important in practicing theinvention but that unspecified materials and conditions are not excludedso long as they do not prevent the benefits of the invention from beingrealized. Exemplary embodiments of the diagnostic methods of theinvention are described above in detail.

In a subject kit, the oncogenic E6 detection reaction may be performedusing an aqueous or solid substrate, where the kit may comprise reagentsfor use with several separation and detection platforms such as teststrips, sandwich assays, etc. In many embodiments of the test strip kit,the test strip has bound thereto a PDZ domain polypeptide thatspecifically binds the PL domain of an oncogenic E6 protein and capturesoncogenic E6 protein on the solid support. The kit usually comprises asubject antibody for detection, which is either directly or indirectlydetectable, and which binds to the oncogenic E6 protein to allow itsdetection. Kits may also include components for conducting western blots(e.g., pre-made gels, membranes, transfer systems, etc.); components forcarrying out ELISAs (e.g., 96-well plates); components for carrying outimmunoprecipitation (e.g. protein A); columns, especially spin columns,for affinity or size separation of oncogenic E6 protein from a sample(e.g. gel filtration columns, PDZ domain polypeptide columns, sizeexclusion columns, membrane cut-off spin columns etc.).

Subject kits may also contain control samples containing oncogenic ornon-oncogenic E6, and/or a dilution series of oncogenic E6, where thedilution series represents a range of appropriate standards with which auser of the kit can compare their results and estimate the level ofoncogenic E6 in their sample. Such a dilution series may provide anestimation of the progression of any cancer in a patient. Fluorescence,color, or autoradiological film development results may also be comparedto a standard curves of fluorescence, color or film density provided bythe kit.

In addition to above-mentioned components, the subject kits typicallyfurther include instructions for using the components of the kit topractice the subject methods. The instructions for practicing thesubject methods are generally recorded on a suitable recording medium.For example, the instructions may be printed on a substrate, such aspaper or plastic, etc. As such, the instructions may be present in thekits as a package insert, in the labeling of the container of the kit orcomponents thereof (i.e., associated with the packaging or subpackaging)etc. In other embodiments, the instructions are present as an electronicstorage data file present on a suitable computer readable storagemedium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actualinstructions are not present in the kit, but means for obtaining theinstructions from a remote source, e.g. via the internet, are provided.An example of this embodiment is a kit that includes a web address wherethe instructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

Also provided by the subject invention are kits including at least acomputer readable medium including programming as discussed above andinstructions. The instructions may include installation or setupdirections. The instructions may include directions for use of theinvention with options or combinations of options as described above. Incertain embodiments, the instructions include both types of information.

The instructions are generally recorded on a suitable recording medium.For example, the instructions may be printed on a substrate, such aspaper or plastic, etc. As such, the instructions may be present in thekits as a package insert, in the labeling of the container of the kit orcomponents thereof (i.e., associated with the packaging orsubpackaging), etc. In other embodiments, the instructions are presentas an electronic storage data file present on a suitable computerreadable storage medium, e.g., CD-ROM, diskette, etc, including the samemedium on which the program is presented.

Utility

The antibodies and methods of the instant invention are useful for avariety of diagnostic analyses. The instant antibodies and methods areuseful for diagnosing infection by an oncogenic strain of HPV in anindividual; for determining the likelihood of having cancer; fordetermining a patient's response to treatment for HPV; for determiningthe severity of HPV infection in an individual; and for monitoring theprogression of HPV in an individual. The antibodies and the methods ofthe instant invention are useful in the diagnosis of infection with anoncogenic or a non-oncogenic strain of HPV associated with cancer,including cervical, ovarian, breast, anus, penis, prostate, larynx andthe buccal cavity, tonsils, nasal passage, skin, bladder, head and necksquamous-cell, occasional periungal carcinomas, as well as benignanogenital warts. The antibodies and the methods of the instantinvention are useful in the diagnosis of infection with an oncogenic ora non-oncogenic strain of HPV associated with Netherton's syndrome,epidermolysis verruciformis, endometriosis, and other disorders. Theantibodies and the methods of the instant invention are useful in thediagnosis of infection with an oncogenic or a non-oncogenic strain ofHPV in adult women, adult men, fetuses, infants, children, andimmunocompromised individuals.

The subject methods may generally be performed on biological samplesfrom living subjects. A particularly advantageous feature of theinvention is that the methods can simultaneously detect, in onereaction, several known oncogenic strains of HPV.

In particular embodiments, the antibodies of the invention may beemployed in immunohistological examination of a sample.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Sequence Analysis of HPV E6 Proteins to Determine OncogenicPotential

PDZ proteins are known to bind certain carboxyl-terminal sequences ofproteins (PLs). PL sequences that bind PDZ domains are predictable, andhave been described in greater detail in U.S. patent application Ser.Nos. 09/710,059, 09/724,553 and 09/688,017. One of the major classes ofPL motifs is the set of proteins terminating in the sequences—X—(S/T)-X-(V/I/L). We have examined the C-terminal sequences of E6proteins from a number of HPV strains, All of the strains determined tobe oncogenic by the National Cancer Institute exhibit a consensus PDZbinding sequence. Those E6 proteins from papillomavirus strains that arenot cancerous lack a sequence that would be predicted to bind to PDZdomains, thus suggesting that interaction with PDZ proteins is aprerequisite for causing cancer in humans. This correlation betweenpresence of a PL and ability to cause cancer is 100% in the sequencesexamined (Table 3A). In theory, with the disclosed PL consensussequences from the patents listed supra, new variants of HPVs can beassessed for their ability to bind PDZ proteins and oncogenicity can bepredicted on the basis of whether a PL is present. Earlier this year,five new oncogenic strains of Human papillomavirus were identified andtheir E6 proteins sequenced. As predicted, these proteins all contain aPL consensus sequence (Table 3B).

TABLE 3A Correlation of E6 PDZ-ligands and oncogenicity E6 C-terminalSeq ID HPV strain sequence PL yes/no Oncogenic No HPV 4 GYCRNCIRKQ No No33 HPV 11 WTTCMEDLLP No No 34 HPV 20 GICRLCKHFQ No No 35 HPV 24KGLCRQCKQI No No 36 HPV 28 WLRCTVRIPQ No No 37 HPV 36 RQCKHFYNDW No No38 HPV 48 CRNCISHEGR No No 39 HPV 50 CCRNCYEHEG No No 40 HPV 16SSRTRRETQL Yes Yes 41 HPV 18 RLQRRRETQV Yes Yes 42 HPV 31 WRRPRTETQV YesYes 43 HPV 35 WKPTRRETEV Yes Yes 44 HPV 30 RRTLRRETQV Yes Yes 45 HPV 39RRLTRRETQV Yes Yes 46 HPV 45 RLRRRRETQV Yes Yes 47 HPV 51 RLQRRNETQV YesYes 48 HPV 52 RLQRRRVTQV Yes Yes 49 HPV 56 TSREPRESTV Yes Yes 50 HPV 59QRQARSETLV Yes Yes 51 HPV 58 RLQRRRQTQV Yes Yes 52 HPV 33 RLQRRRETAL YesYes 53 HPV 66 TSRQATESTV Yes Yes* 54 HPV 68 RRRTRQETQV Yes Yes 55 HPV 69RRREATETQV Yes Yes 56 HPV 34 QCWRPSATVV Yes Yes 356 HPV 67 WRPQRTQTQVYes Yes 357 HPV 70 RRRIRRETQV Yes Yes 358 Table 3A: E6 C-terminalsequences and oncogenicity. HPV variants are listed at the left.Sequences were identified from Genbank sequence records. PL Yes/No wasdefined by a match or non-match to the consenses determined by theinventors and by Songyang et al. -X-(S/T)-X-(V/I/L). Oncogenicity datacollected from National Cancer Institute; Kawashima et al. (1986) J.Virol. 57: 688-692; Kirii et al. (1998) Virus Genes 17: 117-121;Forslund et al. (1996) J. Clin. Microbiol. 34: 802-809. *Only found inoncogenic strains co-transfected with other oncogenic proteins.

TABLE 3B Correlation of recently identified oncogenic E6 proteins E6C-terminal Seq ID HPV strain sequence PL yes/no oncogenic No HPV 26RPRRQTETQV Yes Yes 63 HPV 53 RHTTATESAV Yes Yes 64 HPV 66 TSRQATESTV YesYes 65 HPV 73 RCWRPSATVV Yes Yes 66 HPV 82 PPRQRSETQV Yes Yes 67 Table3B: E6 C-terminal sequences and oncogenicity. HPV variants are listed atthe left. Sequences were identified from Genbank sequence records. PLYes/No was defined by a match or non-match to the consensus sequence:-X-(S/T)-X-(V/I/L). Oncogenicity data on new strains collected from NEngl J Med 2003; 348: 518-527.

These tables provide a classification of the HPV strains based on thesequence of the C-terminal four amino acids of the E6 protein encoded bythe HPV genome. The 21 oncogenic strains of HPV fall into one of 11classes (based on the C-terminal four amino acids), and HPV strains notspecifically listed above may also fall into these classes. As such, itis desirable to detect HPV strains from all 11 classes: the instantmethods provide such detection.

A cross-reactive antibodie of the invention may recognize E6 proteinsfrom HPV strains of multiple (e.g., 2, 3, 4, 5, 6, or 7 or moredifferent) classes.

Example 2 Identification of PDZ Domains that Interact with the C-Terminiof Oncogenic E6 Proteins

In order to determine the PDZ domains that can be used to detectoncogenic E6 proteins in a diagnostic assay, the assay was used toidentify interactions between E6 PLs and PDZ domains. Peptides weresynthesized corresponding to the C-terminal amino acid sequences of E6proteins from oncogenic strains of human papillomavirus. These peptideswere assessed for the ability to bind PDZ domains using an assay and PDZproteins synthesized from the expression constructs described in greaterdetail in U.S. patent application Ser. Nos. 09/710,059, 09/724,553 and09/688,017. Results of these assays that show a high binding affinityare listed in Table 4 below.

As we can see below, there a large number of PDZ domains that bind someof the oncogenic E6 proteins and the second PDZ domain from MAGI-1 bindsall of the oncogenic E6 PLs tested. The PDZ domain of TIP-1 binds allbut one of the oncogenic E6 PLs tested, and may be useful in conjunctionwith MAGI-1 domain 2 for detecting the presence of oncogenic E6proteins.

In a similar manner, peptides corresponding to the C-terminal ends ofseveral non-oncogenic E6 proteins were tested with assay. None of thepeptides showed any affinity for binding PDZ domains.

TABLE 4 higher affinity interactions between HPV E6 PLs and PDZ domainsPDZ binding partner HPV strain (signal 4 and 5 of 0-5) HPV 35 Atrophin-1interact. prot. (T E V) (PDZ # 1, 3, 5) Magi1 (PDZ # 2, 3, 4, 5) Lim-RilFLJ 11215 MUPP-1 (PDZ #10) KIAA 1095 (PDZ #1) PTN-4 INADL (PDZ #8)Vartul (PDZ # 1, 2, 3) Syntrophin-1 alpha Syntrophin gamma-1 TAX IP2KIAA 0807 KIAA 1634 (PDZ #1) DLG1 (PDZ1, 2) NeDLG (1, 2, 3,) Sim. Ratouter membrane (PDZ #1) MUPP-1 (PDZ #13) PSD 95 (1, 2, 3) HPV 58Atrophin-1 interact. prot. (PDZ # 1) (T Q V) Magi1 (PDZ #2) DLG1 (PDZ1,2) DLG2 (PDZ #2) KIAA 0807 KIAA 1634 (PDZ #1) NeDLG (1, 2) Sim. Ratouter membrane (PDZ #1) PSD 95 (1, 2, 3) INADL (PDZ #8) TIP-1 HPV 16*TIP-1 (T Q L) Magi1 (PDZ #2) HPV 18* TIP1 (T Q V) Magi 1 (PDZ #2) HPV 33Magi1 (PDZ #2) (T A L) TIP1 DLG1 Vartul (PDZ #1) KIAA 0807 KIAA 1095(Semcap3) (PDZ #1) KIAA 1934 (PDZ #1) NeDLG (PDZ #1, 2) Rat outermembrane (PDZ #1) PSD 95 (PDZ #3 and 1-3) HPV 66 DLG1 (PDZ #1, 2) (S TV) NeDLG (PDZ #2) PSD 95 (PDZ #1, 2, 3) Magi1 (PDZ #2) KIAA 0807 KIAA1634 (PDZ #1) DLG2 (PDZ #2) Rat outer membrane (PDZ #1) NeDLG (1, 2)TIP-1 HPV 52 Magi1 (PDZ #2) (T Q V) Table 4: Interactions between the E6C-termini of several HPV variants and human PDZ domains. HPV straindenotes the strain from which the E6 C-terminal peptide sequenceinformation was taken. Peptides used in the assay varied from 18 to 20amino acids in length, and the terminal four residues are listed inparenthesis. Names to the right of each HPV E6 variant denote the humanPDZ domain(s) (with domain number in parenthesis for proteins withmultiple PDZ domains) that saturated binding with the E6 peptide inassay *denotes that the PDZ domains of hDlg1 were not tested againstthese proteins yet due to limited material, although both have beenshown to bind hDlg1 in the literature.

The subject antibodies may be used with these oncogenic HPV E6-bindingPDZ proteins in methods of detecting oncogenic strains of HPV.

Materials and Methods for Examples 3-7

Immunization protocol: Five 8 week-old female BALB/c mice are immunizedintraperitoneally, in the footpad, or subcutaneously on day zero with 20μg of MBP-E6 fusion protein or 100 μg of E6 conjugated-peptide and 20 μgof polyI/polyC polymer or complete Freund's adjuvant. Animals areboosted with 20 μg of E6 protein and polyI/polyC or incomplete Freund'sadjuvant. Test bleeds are performed 3 days after the last boostandscreened by ELISA against the corresponding E6 protein. Immunoreactivemice have a final boost three days prior to fusion.

ELISA screening of serum antibody titer and B cell hybridomasupernatants: ELISA plates are coated with appropriate fusion protein,washed, and blocked with PBS containing 2% BSA (Sigma). Then the testsample (immune sera or hybridoma supernatant) is added, along with apre-immune or irrelevant supernatant negative control. After incubationthe plate is washed and anti-mouse IgG-HRP conjugate (JacksonLaboratories) in PBS/2% BSA is added. After thorough washing, TMBsubstrate is added for 30 minutes, followed by termination of thereaction with 0.18 M H₂SO₄. The plate is then read at 450 nm using aMolecular Devices' THERMO Max microplate reader.

Fusion: On the day of fusion, the animals are sacrificed, bloodcollected, and the spleens excised and minced with scissors. The cellsare then gently teased up and down with a pipette, filtered through asterile 70 μm nylon filter and washed by centrifugation. Splenocytes andthe FOX-NY myeloma partner (maintained prior to fusion in log growth)are resuspended in serum-free-RPMI medium, combined at a ratio of 4:1and spun down together. The fusion is then performed by adding 1 ml of50% PEG (Sigma) drop-wise over one minute, followed by stirring thecells for one minute. Then 2 ml of RPMI/15% FCS media is added drop-wiseover two minutes, followed by 8 ml of RPMI/15% FCS over 2 minutes withconstant stirring. This mixture is centrifuged, and the cells are gentlyresuspended at 10⁸ cells/ml in RPMI/15% FCS+1×HAT media (Sigma) andplated out in 96-well flat bottom plates at 200 μl/well. After 5 days˜100 μl old medium is replaced by aspirating out of wells, and adding100 μl fresh RPMI/HAT medium. Hybridomas are kept in RPMI/HAT for ˜7days. Then are grown in RPMI/15% FCS containing 1×HT for ˜1 week. Wellsare assayed for antibody activity by ELISA when they are 10-30%confluent.

Hybridoma cloning, antibody purification and isotyping: Wells whosesupernatants give the desired activity were selected for cloning. Cellsare cloned by limiting dilution in a 96-well flat bottom plate.Purification of antibodies from tissue culture supernatants is performedby protein G and A affinity chromatography (Amersham). The isotype ofthe antibodies is determined using Cytometric bead array.

Cell lines: Cervical cancer cell lines expressing listed strains of HPVE6 were purchased from ATCC, and are shown in the following table:

ATCC Common E6 GenBank Name Name Organism Tissue type Accession # HTB-31C-33A human cervix None HTB-32 HT-3 human cervix 30 HTB-33 ME-180 humancervix 68b M73258 HTB-34 MS751 human cervix 45 X74479 HTB-35 SiHa humancervix 16 CRL-1550 CaSki human cervix 16 CRL-1594 C-41 human cervix 18CRL-1595 C-4-II human cervix 18

Stably or transiently transfected cells were produced using thefollowing methods:

The following stable cell lines were made: 3A-HA-E6-26 (expresses HPV 26E6); C33A-HA-E6-53 (expresses HPV 53 E6); C33A-HA-E6-58 (expresses HPV58 E6); C33A-HA-E6-59 (expresses HPV 59 E6); C33A-HA-E6-66 (expressesHPV 66 E6); C33A-HA-E6-69 (expresses HPV 69 E6) and C33A-HA-E6-73(expresses HPV 73 E6).

Calcium Phosphate Transfection of Mammalian Cell Lines

Materials: Deionized water, 2M CaCl₂, 2×HBS pH 7.1, 25 mM Chloroquine(1000×), DNA.

Day 0: Plate 0.8 million cells in each well of a 6-well plate the nightbefore transfection. (2 wells for each construct, therefore, 3constructs in a 6-well plate)

Day 1: a) Aliquot appropriate cell media and add Chloroquine (Add 12.5μl for every 10 ml of media. The extra 2.5 μl is to account for the 500ul of the calcium phosphate+DNA solution that will be added to the cellslater). b) Aspirate media off the cells and add 2 mL of themedia+Chloroquine solution. Return cells to incubator. c) In a 5 mLpolypropylene tube, add the following in the order listed: i) deionizedwater, ii) DNA and iii) 2M CaCl₂ as follows:

DNA Deionized water 2M CaCl2 2X HBS 10 μg (DNA + 64 + dH20 = 500 μl 64μl 500 μl

d) Add 500 μl of the DNA mix drop wise to the 2×HBS while bubbling withautomatic pipetman and Pasteur pipette; e) Add 500 μlDNA/calcium/phosphate solution to each well; and f) Incubate inincubator for 8 hours, then replace media with normal growth media.

Day 3: Start selection with G-418 (Gibco) at 1 mg/ml

Cells for transient expression of HPV 51 E6 were produced by standardmethods.

Example 3 HPV-E6 Recombinant Protein Expression and Purification

Polynucleotides encoding E6 proteins of high-risk HPV types listed abovewere chemically synthesized (DNA 2.0, Menlo Park, Calif.) or cloned viaRT-PCR from cervical cancer cell lines. Both maltose-binding-protein-E6(MBP-E6) and glutathione-S-transferase-E6 (GST-E6) fusion protein typeswere used. Production of GST-E6 and MBP-E6 proteins were by standardprotocols recommended by the suppliers (Amersham and New EnglandBiolabs, respectively). Proteins were expressed in DH5α E. coli usingIPTG driven induction. A 2 h induction at 37° C. yielded GST-E6 orMBP-E6 recombinant proteins at ˜1 mg/L, whereas induction overnight at20° C. and purification including rebinding of protein to the gel matrixresulted in final yield of 2-10 mg/L. Purity of MBP-E6 proteins wasestimated to be >90% based on PAGE analysis. Recombinant E6 fusionproteins were used as immunogens.

Example 4 Immunization, Fusion, Screening and Cloning of HybridomasSecreting Monoclonal Antibodies Against E6 Protein

Mice were immunized with each of the HPV E6 proteins. A variety ofimmunization protocols including varying antigen doses (100 μg-10 μg),adjuvants (CFA/IFA, poly(I)-poly(C), CpG+Alum) and routes (subcutaneous,intraperitoneal) were tested. A service facility for animal care,handling of immunizations and sera collection was contracted (Josman,Napa, Calif.). Immunization projects were set up with 5-15 mice each.Sera of immunized mice were tested in ELISA against the recombinant E6protein. Mice showing sufficiently high titers (OD above 1 at 1:1000dilution) against E6 in their sera were selected for fusions.

To increase the frequency of hybridomas secreting of anti-E6 antibodies,the recombinant E6 protein used in the final boost contained a differenttag from that used during the immunization (GST-E6 was used in the boostwhen immunizations occurred with MBP-E6, and vice versa)

Example 5 Spleen Cells of Selected Mice were Fused

Hybridoma supernatants were tested via direct antigen ELISA against theMBP-E6 used in the immunization and MBP protein as negative control.Supernatants that showed reactivity for MBP-E6 (immunogen) but not forMBP were selected for further analysis. Selected supernatants weretested further by slot western blot for reactivity against recombinantMBP-E6 and GST-E6, to reconfirm presence of anti-E6 mAb. At this stage,hybridomas were cloned by limiting dilution to isolate hybridoma clonessecreting anti-E6 mAb.

To further characterize the reactivity of the hybridomas, selectedsupernatants were tested in an ELISA against the recombinant E6proteins, as well as GST-INADL (PDZ) and GST-MAGI1-PDZ1 that served asnegative controls. GST-1NADL represents a class of proteins that, whenpurified in prokaryotic expression systems, tend to be associated with abacterial contaminating that are also present in the MBP-/GST-E6 proteinpreparations used for immunizations. This control ensured thatreactivity found in supernatants reflected a mAb binding to HPV-E6, andnot against the associated contaminants.

Example 6 Cross-Reactivity Pattern of ANTI-E6 Monoclonal Antibodies

The cross-reactivity pattern of anti-E6 mAbs against E6 proteins otherthan the one used as immunogen was tested. For this E6 panel test, adirect ELISA approach is used (recombinant E6 protein is coated on theplate).

Monoclonal antibodies against the E6 protein of high-risk HPV types thatcause cervical cancer (e.g., HPV 16, 18, 26, 30, 31, 34, 45, 51, 52, 53,58, 59, 66, 68b, 69, 70, 73, 82) were produced.

A summary of results showing cross-reactivity of the antibodies producedis shown In Table 5 below.

TABLE 5 Endogenous mAb HPV-E6 type binding HPV-E6 binding E6 detectionImmunogen and Immunization route and designation profile—direct ELISAprofile S2 ELISA S2 ELISA boosts/last boost adjuvant F12-1B9 18, 45, 66N.D. N.D. HPV18-[MBP]-E6/ subcutaneous/Adjuvant: F12-1C9 18 N.D. N.D.HPV18-[GST]-E6 complete/incomplete F12-1H12 18 N.D. N.D. Freund's(initial/follow up F12-2D2 18, 45, 66 N.D. N.D. injections) F12-3B2 18N.D. N.D. F12-3D5 18, 45, 66, 82 18, 45 18, 45 F12-4A11 18 18 18 F12-4E2 18 18, 45 N.D. F12-5C2 18 N.D. N.D. F12-6D9 18, 45 N.D. N.D. F12-6F518 N.D. N.D. F12-6F6 18, 45 N.D. N.D. F12-6H2 18, 45, 66, 82 N.D. N.D.F12-7A10 18, 45 N.D. N.D. F12-7F10 18 N.D. N.D. F12-8A3 18, 45 N.D. N.D.F12-8B8 18 N.D. N.D. F16-4H12 16, 35 16 N.D. HPV16-[MBP]-E6/subcutaneous/Adjuvant: F16-5D5 16, 35 16 does not HPV16-[GST]-E6complete/incomplete recognize 16 Freund's (initial/follow up injections)F17-1 E11 26, 51, 52, 53, 58 N.D. N.D. HPV58-[MBP]-E6/subctaneous/Adjuvant: F17-6G9 33, 58 58 does not HPV58-[GST]-E6complete/incomplete recognize 58 Freund's (initial/follow up injections)F18-3G11 16 16 does not HPV16-[GST]-E6/ subcutaneous/Adjuvant: recognize16 HPV16-[MBP]-E6 complete/incomplete F18-4C9 16 N.D. N.D. Freund's(initial/follow up F18-5H3 16 N.D. N.D. injections) F18-7H8 16 N.D. N.D.F18-8G11 16 N.D. N.D. F18-9B10 16, 73 N.D. N.D. F18-10 E6 16 16 16F18-10 E10 16 N.D. N.D. F19-6D10 18, 68b 18, 68b does not DNA plasmidrecognize 18 or immunization; boost 68b with HPV18-E6 (MBP- F19-6F9 18,68b 18, 68b N.D. E6/GST-E6) F19-7B12 18, 35, 68b N.D. N.D. F19-7C7 18,68b N.D. N.D. F19-8E2 18, 35, 68b N.D. N.D. F20-2H5 16, 18, 35, 45 18,35, 45 does not HPV45-[MBP]-E6/ subcutaneous/Adjuvant: recognize 18 orHPV45-[GST]-E6 complete/incomplete 45; 35 N.D. Freund's (initial/followup injections) F21-1D12 18, 30, 52, 58 30, 58 does not HPV58-[MBP]-E6/footpad injection/Adjuvant: recognize 30 or HPV58-[GST]-E6 CpG-ALUM 58F21-3A3 18, 58 N.D. N.D. F21-3H2 18, 30, 52, 58 58 N.D. F21-4 E10 18,30, 52, 58 58 N.D. F21-4F9 18, 33, 58 N.D. N.D. F21-4H1 18, 30, 33, 52,58 33, 58 33, 58 F21-5B2 16, 18, 30, 52, 58, 59, 68b N.D. N.D. F22-1C1226, 51, 69 51 51 HPV51-[MBP]-E6/ subctaneous/Adjuvant: F22-10D11 26, 30,31, 35, 51, N.D. N.D. HPV51-[GST]-E6 complete/incomplete 53, 66, 69, 82Freund's (initial/follow up F22-10F10 26, 51, 69 51 N.D. injections)F24-2D6 26, 51, 69, 82 26, 69 26, 69 HPV69-[MBP]-E6/subcutaneous/Adjuvant: F24-4B12 26, 51, 53, 69, 73, 82 N.D. N.D.HPV69-[GST]-E6 complete/incomplete F24-4F2 26, 51, 69, 82 26, 69, 82 26,69; 82 N.D. Freund's (initial/follow up F24-4G1 26, 51, 69, 82 N.D. N.D.injections) F24-8H12 26, 51, 69, 82 26, 69, 82 N.D. F24-9H12 26, 51, 69,82 26, 69 N.D. F25-2D11 73 N.D. N.D. HPV73-[MBP]-E6/subcutaneous/Adjuvant: F25-3D10 53, 73, 82 N.D. N.D. HPV73-[GST]-E6complete/incomplete F25-3 E5 16, 34, 59, 70, 73 N.D. N.D. Freund's(initial/follow up F25-4C11 16, 34, 59, 70, 73 34 does not injections)recognize 73, 34 N.D. F26-1B10 51, 53 N.D. N.D. HPV53-[MBP]-E6/subcutaneous/Adjuvant: F26-1B11 53 N.D. N.D. HPV53-[GST]-E6complete/incomplete F26-1D9 53 N.D. N.D. Freund's (initial/follow upF26-1D11 53 N.D. N.D. injections) F26-2B12 53 N.D. N.D. F26-2G5 53 N.D.N.D. F26-3A8 30, 53, 66 N.D. N.D. F26-5H5 53 N.D. N.D. F26-6D10 53 N.D.N.D. F26-8B7 53 N.D. N.D. F26-8H9 53 N.D. N.D. F26-9C2 53 N.D. N.D.F26-9C7 53 N.D. N.D. F26-9D8 53 N.D. N.D. F26-9G5 53, 73, 82 N.D. N.D.F27-3A4 59 N.D. N.D. HPV59-[MBP]-E6/ subcutaneous/Adjuvant:HPV59-[GST]-E6 complete/incomplete Freund's (initial/follow upinjections) 6F4 16 16, 35, 69 recognizes 16 HPV16- poly-I/poly-Cadjuvant/three and 69, 35 N.D. [GST]E6/HPV16- immunizations [GST]E6 4C616 16 N.D. HPV16- poly-I/poly-C adjuvant/three [GST]E6/HPV16-immunizations [GST]E6 3F8 16 16, 35, 51, N.D. HPV16-[MBP]E6-C-poly-I/poly-C adjuvant/three 82, 31, 33 terminal immunizations and 58portion/HPV16- [MBP]E6-C-terminal portion

FIG. 3 shows results obtained from a slot western blot of recombinant E6protein, probed with hybridoma supernatants.

Example 7 Selection of Antibodies for HPV Diagnostic Test

Supernatants from hybridomas reacting with E6 proteins are testedtogether with the oncogenic PL detector in a sandwich ELISA withrecombinant E6 fusion protein.

Monoclonal antibodies are tested in HPV diagnostic ELISA for theirability to detect E6 from cervical cancer cell lines or cellstransfected with E6 (if cell lines are unavailable).

It is evident from the above results and discussion that the subjectinvention provides an important new means for detecting HPV E6 proteins.In particular, the subject invention provides a system for detectingoncogenic strains of HPV. It is superior to current methods because thePDZ protein isolates the oncogenic E6 protein from other analytes of acomplex biological sample, and the protein is detected using an antibodythat cross-reacts with more than one E6 protein. The specificity ofdetection lies in the PDZ protein and the antibody does not need to bindonly oncogenic E6 proteins, as currently required in conventionalmethods. Accordingly, the subject methods and systems find use in avariety of different diagnostic applications. Accordingly, the presentinvention represents a significant contribution to the art.

1. A monoclonal antibody that specifically binds to a sub-sequence of acommon sequence of at least two E6 proteins of HPV strains 16, 18, and45, wherein the common sequence is an amino acid sequence motif that isgenerally conserved between the at least two E6 proteins.
 2. Themonoclonal antibody of claim 1, wherein the antibody specifically bindsto an E6 peptide having SEQ ID NO: 7, 8, or
 60. 3. The monoclonalantibody of claim 1, wherein the antibody specifically binds to an E6peptide having SEQ ID NO: 9, 10, or
 61. 4. The monoclonal antibody ofclaim 1, wherein the antibody specifically binds to an E6 peptide havingSEQ ID NO: 11, 12, or
 62. 5. The monoclonal antibody of claim 1, whereinthe antibody specifically binds to an HPV E6 peptide having SEQ ID NO:1, 2, 3, 4, 5, 6, 57, 58, or
 59. 6. The monoclonal antibody of claim 1,wherein the antibody is cross-reactive with at least three humanpapilloma virus E6 proteins.
 7. The monoclonal antibody of claim 1,wherein the antibody is cross-reactive with at least four humanpapilloma virus E6 proteins.
 8. The monoclonal antibody of claim 1,wherein the antibody has a binding affinity of less than 10⁻⁷ M.
 9. Themonoclonal antibody of claim 1, wherein the antibody has a bindingaffinity of less than 10⁻⁸ M.
 10. The monoclonal antibody of claim 1,wherein the antibody has a binding affinity of less than 10⁻⁹ M.
 11. Anantibody composition comprising a mixture of monoclonal antibodies thatspecifically bind to E6 proteins of HPV strains 16, 18, 31, 33 and 45,wherein at least one of said monoclonal antibodies is cross-reactivewith at least two HPV E6 proteins, wherein the antibody specificallybinds to an E6 peptide comprising a sub-sequence of a common sequence ofat least two E6 proteins of HPV strains 16, 18, and 45, wherein thecommon sequence is an amino acid sequence motif that is generallyconserved between the at least two E6 proteins.
 12. The antibodycomposition of claim 11, wherein at least one of said monoclonalantibodies specifically binds to an E6 peptide having SEQ ID NO: 1, 2,3, 4, 5, 6, 57, 58, or
 59. 13. The antibody composition of claim 11,wherein said mixture of monoclonal antibodies specifically bind to E6proteins of HPV strains 16, 18, 31, 33, 45, 52 and
 58. 14. The antibodycomposition of claim 11, wherein said mixture of monoclonal antibodiesspecifically bind to E6 proteins of HPV strains 16, 18, 31, 33, 45, 52,58, 35 and
 59. 15. A diagnostic kit for the detection of an HPV E6polypeptide in a sample, comprising: the antibody composition of claim11.
 16. The diagnostic kit of claim 15, wherein said monoclonalantibodies are labeled.
 17. The diagnostic kit of claim 15, furthercomprising a PDZ domain polypeptide that binds to an oncogenic HPV E6polypeptide in a sample.
 18. A method of detecting an HPV E6 protein ina sample, comprising: contacting an antibody composition of claim 11with said sample; and detecting any binding of said antibody to saidsample; wherein binding of said antibody to said sample indicates thepresence of an HPV E6 protein.
 19. The method of claim 18, wherein saidsample is suspected of containing an oncogenic strain of HPV.
 20. Amethod of detecting the presence of an oncogenic HPV E6 protein in asample, said method comprising: contacting a sample with a PDZ domainpolypeptide; and detecting any binding of said oncogenic HPV E6 proteinin said sample to said PDZ domain polypeptide using an antibodycomposition of claim 11; wherein binding of said oncogenic HPV E6protein to said PDZ domain polypeptide indicates the presence of anoncogenic HPV E6 protein in said sample.
 21. A system for detecting thepresence of an oncogenic HPV E6 polypeptide in a sample, said systemcomprising: a first and a second binding partner for an oncogenic HPV E6polypeptide, wherein said first binding partner is a PDZ domain proteinand said second binding partner is an antibody of claim
 1. 22. Thesystem of claim 21, wherein at least one of said binding partners isattached to a solid support.